Automated Set-Up for Cell Sorting

ABSTRACT

Apparatus and methods are described for automatically performing set-up steps for flow cytometry operations. The invention provides for the spatial determination of a flow stream and the subsequent automatic alignment of analysis devices and/or collection vessels. The automatic determination of flow stream properties provides for the automatic configuration flow cytometer parameters.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/811,465filed Apr. 12, 2013, the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

Flow cytometers known in the art are used for analyzing and sortingparticles in a fluid sample, such as cells of a blood sample orparticles of interest in any other type of biological or chemicalsample. A flow cytometer typically includes a sample reservoir forreceiving a fluid sample, such as a blood sample, and a sheath reservoircontaining a sheath fluid. The flow cytometer transports the particles(hereinafter called “cells”) in the fluid sample as a cell stream to aflow cell, while also directing the sheath fluid to the flow cell.

Within the flow cell, a liquid sheath is formed around the cell streamto impart a substantially uniform velocity on the cell stream. The flowcell hydrodynamically focuses the cells within the stream to passthrough the center of a laser beam in a flow cell. The point at whichthe cells intersect the laser beam is commonly known as theinterrogation point. As a cell moves through the interrogation point, itcauses the laser light to scatter. The laser light also excitescomponents in the cell stream that have fluorescent properties, such asfluorescent markers that have been added to the fluid sample and adheredto certain cells of interest, or fluorescent beads mixed into thestream. The flow cytometer includes an appropriate detection systemconsisting of photomultiplier tubes, photodiodes or other lightdetecting devices, which are focused at the intersection point. The flowcytometer analyzes the detected light to measure physical andfluorescent properties of the cell. The flow cytometer can further sortthe cells based on these measured properties. The flow stream exits theflow cell via a nozzle with a nozzle diameter that is appropriate forthe fluidics system and sort rate desired.

To sort cells by an electrostatic method, the desired cell must becontained within an electrically charged droplet. To produce droplets,the flow cell is rapidly vibrated by an acoustic device, such as apiezoelectric element. The volume of a droplet is conventionallyestimated by the hydrodynamic properties of the flow stream and thenozzle dimensions. To charge the droplet, the flow cell includes acharging element whose electrical potential can be rapidly changed.Because the cell stream exits the flow cell in a substantially downwardvertical direction, the droplets also propagate in that direction afterthey are formed. Droplets, whether they are charged or are unchargedmust be collected in a sample collection vessel that is appropriatelydirected to collect the one or more flow streams generated by thedeflection plates. Accordingly, the droplets and the cells containedtherein may be collected in appropriate collection vessels downstream ofthe plates.

Known flow cytometers similar to the type described above are described,for example, in U.S. Pat. Nos. 3,960,449, 4,347,935, 4,667,830,5,464,581, 5,483,469, 5,602,039, 5,643,796 and 5,700,692, the entirecontents of each patent being incorporated by reference herein. Othertypes of known flow cytometer are the FACSVantage™, FACSort™,FACSCount™, FACScan™, and FACSCalibur™ systems, each manufactured byBecton Dickinson and Company, the assignee of the present invention.

Although this method generally enables the flow cytometer to dispensesorted cells into collection vessels and therefore sort the cells ofinterest with reasonable accuracy, the method requires a substantialamount of user input at the time of set-up. The flow stream andcollection vessels are conventionally manually aligned. The fluidicsparameters such as flow rate and sheath fluid composition must bematched with an appropriate nozzle diameter.

SUMMARY

Aspects of the present disclosure include systems for adjusting one ormore parameters of a flow cytometer. Systems according to certainembodiments include an imaging sensor configured to capture one or moreimages of a detection field of a flow stream of the flow cytometer and aprocessor configured to generate a data signal from the one or morecaptured images such that the system adjusts one or more parameters ofthe flow cytometer in response to the data signal.

In certain embodiments, the subject systems are configured to reduce theneed for user input or manual adjustment during sample analysis with aflow cytometer. In some embodiments, systems of interest may bepartially or fully automated so that adjustments to parameters of a flowcytometer are processor controlled. In certain embodiments, the subjectsystems are configured to adjust one or more parameters of the flowcytometer without any human input.

In certain embodiments, the present disclosure provides a system forautomatically localizing a stream position in a liquid flow from a flowcytometer comprising a first camera, adapted to detect a stream positionin a first detection field and to generate a first signal representativeof the stream position and a first stage wherein the first stage isoperationally connected to the first camera and configured to move in anXY plane in response to the first signal.

The system may further include a second camera adapted to detect a steamposition in a second detection field and to generate a second signalrepresentative of the stream position wherein the first and seconddetection fields of the first and second cameras are substantiallyorthogonally oriented in the XY plane wherein the first stage isoperationally connected to the second camera and configured to move theXY plane in response to the second signal in addition to the firstsignal. In some embodiments a laser is mounted or collection device ismounted on the first stage.

In some embodiments the system may include a second stage wherein acollection device is mounted on the second stage and the second stage isconfigured to move in the XY plane in response to the first signal andthe second signal. The system may further comprise an electrical systemconfigured to adjust an electrical charge on the flow stream in responseto the second signal from the second camera. The operational connectionbetween the cameras and the stages may be mediated by a processorconnected to the first camera and the first and second camera and thefirst stage and wherein the processor is configured to receive thesignals from the first and second cameras and calculate an optimumposition for the first stage. In some embodiments the operationalconnection is mediated by a processor connected to the first and secondcamera and the second stage and configured to receive the signals fromthe first and second cameras and calculate an optimum position for thesecond stage. In some embodiments the stream may include a series ofdrops.

A system according to certain embodiments is provided for automaticallydetermining a nozzle opening diameter with a first camera, adapted todetect a stream dimension in a first detection field and to generate afirst signal representative of the stream dimension and a processorhaving memory with instructions thereon configured to determine a valuefor the nozzle opening diameter from the stream dimension and transmitthe value to a flow cytometer. The stream dimension may be the width ofthe stream. In some embodiments the flow cytometer may be configured toautomatically adjust a series of parameters after receiving thetransmitted value. The series of parameters may be selected from thegroup comprising hydrostatic pressure, drop charge, deflection voltage,charge correction value, drop delay, drop frequency, drop amplitude, andcharge phase.

Aspects of the disclosure also include methods for adjusting one or moreparameters of a flow cytometer. Methods according to certain embodimentsinclude capturing one or more images of a flow stream of the flowcytometer in a detection field, determining one or more properties ofthe flow stream in the detection field, generating a data signalcorresponding to the one or more properties of the flow stream andadjusting one or more parameters of the flow cytometer in response tothe data signal.

Aspects of the present disclosure also include computer controlledsystems for practicing the subject methods, where the systems furtherinclude one or more computers having processors configured to automateone or more steps of the methods described herein. In some embodiments,systems include a computer having a computer readable storage mediumwith a computer program stored thereon, where the computer program whenloaded on the computer includes instructions for capturing one or moreimages of a flow stream of the flow cytometer in a detection field;algorithm for determining the spatial position of the flow stream in thedetection field; algorithm for generating a data signal corresponding tothe spatial position of the flow stream; and instructions for adjustingone or more parameters of the flow cytometer in response to the datasignal. In certain instances, systems include a computer having acomputer readable storage medium with a computer program stored thereon,where the computer program when loaded on the computer includesinstructions for capturing one or more images of a flow stream of theflow cytometer in a detection field; algorithm for determining thephysical dimensions of the flow stream in the detection field; algorithmfor generating a data signal corresponding to the physical dimensions ofthe flow stream; and instructions for adjusting one or more parametersof the flow cytometer in response to the data signal.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 depicts a schematic illustration of a system according to certainembodiments.

FIG. 2 depicts a flow chart illustrating steps for practicing methods ofthe present disclosure according to certain embodiments.

FIG. 3 depicts a flow chart illustrating steps for practicing methods ofthe present disclosure according to certain embodiments.

DETAILED DESCRIPTION

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, the present disclosure provides systems configuredto automate adjustments of one or more parameters of a flow cytometer.In further describing embodiments of the disclosure, systems configuredto adjust one or more parameters of a flow cytometer are first describedin greater detail. Next, methods for adjusting one or more parameters ofa flow cytometer with the subject systems are described. Computercontrolled systems which automate adjustments to one or more parametersof a flow cytometer are also provided.

Systems for Adjusting Parameters of a Flow Cytometer

Aspects of the present disclosure include systems configured to adjustparameters of a flow cytometer. The term “adjusting” is used herein inits conventional sense to refer to changing one or more functionalparameters of the flow cytometer. As described in greater detail below,the desired adjustment may vary in terms of goal, where in someinstances the desired adjustments are adjustments that ultimately resultin enhanced efficiency of some desirable parameter, e.g., improved cellsorting accuracy, enhanced particle collection, identifying componentmalfunction (e.g., clogged flow cell nozzle), energy consumption,particle charging efficiency, more accurate particle charging, enhancedparticle deflection during cell sorting, among other adjustments. Inembodiments, the subject systems are configured to reduce the need foruser input or manual adjustment during sample analysis with a flowcytometer. In certain embodiments, systems of interest may be fullyautomated so that adjustments to parameters of a flow cytometer areprocessor controlled. By “fully automated” is meant that adjustmentsmade in response to data signals corresponding to one or more parametersof the flow stream and derived from one or more captured images of theflow stream requires little to no human intervention or manual inputinto the subject systems. In certain embodiments, the subject systemsare configured to adjust one or more parameters of the flow cytometerbased on the data signals corresponding to one or more parameters of theflow stream without any human intervention.

As summarized above, systems include one or more imaging sensorsconfigured to capture images of a flow cytometer flow stream in one ormore detection fields. By “detection field” is meant the region of theflow stream which is imaged by the one or more imaging sensors.Detection fields may vary depending on the properties of the flow streambeing interrogated. In embodiments, the detection field may span 0.001mm or more of the flow stream, such as 0.005 mm or more, such as 0.01 mmor more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mmor more, such as 1 mm or more, such as 2 mm or more, such as 5 mm ormore and including 10 mm or more of the flow stream. For example, wherethe subject systems are configured to determine a physical dimension(e.g., width) of the flow stream, the detection field may be a planarcross-section of the flow stream. In another example, where the subjectsystems are configured to determine the spatial position of the flowstream, the detection field may be a predetermined length of the flowstream, such as for example to determine the angle made by the flowstream with respect to the axis of the flow cell nozzle.

The detection field interrogated by the subject systems may varydepending on the parameter of the flow cytometer being adjusted. In someembodiments, the detection field includes the flow cell nozzle orifice.In other embodiments, the detection field includes the location of theflow stream where the drops containing the particles of interest arecharged (i.e., the “break-off” point where the continuous flow streambegins to form discrete droplets). In yet other embodiments, thedetection field includes the region where charged particles aredeflected by deflector plates during cell sorting.

Systems include one or more imaging sensors configured to capture imagesof a flow stream in a detection field. The imaging sensor may be anysuitable device capable of capturing and converting an optical imageinto an electronic data signal, including but not limited tocharge-coupled devices, semiconductor charge-coupled devices (CCD),active pixel sensors (APS), complementary metal-oxide semiconductor(CMOS) image sensors or N-type metal-oxide semiconductor (NMOS) imagesensors. In some embodiments, the imaging sensor is a CCD camera. Forexample, the camera may be an electron multiplying CCD (EMCCD) camera oran intensified CCD (ICCD) camera. In other embodiments, the imagingsensor is a CMOS-type camera.

Depending on the number of detection fields being interrogated and flowcytometer parameters of interest, the number of imaging sensors in thesubject systems may vary, as desired. For example, the subject systemsmay include one imaging sensor or more, such as two imaging sensors ormore, such as three imaging sensors or more, such as four imagingsensors or more, such as five imaging sensors or more and including tenimaging sensors or more. In certain embodiments, systems include oneimaging sensor. In other embodiments, systems include two imagingsensors. Where systems include more than one imaging sensor, eachimaging sensors may be oriented with respect to the other (as referencedin an X-Y plane) at an angle ranging from 10° to 90°, such as from 15°to 85°, such as from 20° to 80°, such as from 25° to 75° and includingfrom 30° to 60°. In certain embodiments, each imaging sensor is orientedorthogonally (as referenced in an X-Y plane) to each other. For example,where the subject systems include two imaging sensors, the first imagingsensor is oriented orthogonally (as referenced in an X-Y plane) to thesecond imaging sensor.

Where the subject systems include more than one imaging sensor, eachimaging sensor may be the same or a combination of sensors. For example,where the subject systems include two imaging sensors, in someembodiments the first imaging sensor is a CCD-type device and the secondimaging sensor is a CMOS-type device. In other embodiments, both thefirst and second imaging sensor are CCD-type devices. In yet otherembodiments, both the first and second imaging sensors are CMOS-typedevices.

In some embodiments, the imaging sensors are stationary, maintaining asingle position within the flow cytometer. In other embodiments, theimaging sensors may be configured to move along the path of the flowstream. For instance, the imaging sensor may be configured to moveupstream and downstream alongside the flow stream capturing images in aplurality of detection fields. For example, systems may include animaging sensor which is adapted to capture images in two or moredifferent detection fields along the flow stream, such as 3 or moredetection fields, such as 4 or more detection fields and including 5 ormore detections fields. Where the imaging sensor is configured to movealong the flow stream, the imaging sensor may be moved along the flowstream path continuously or in discrete intervals. In some embodiments,the imaging sensor is displaced continuously. In other embodiments, theimaging sensor may be displaced along the flow stream path in discreteintervals, such as for example in 1 mm or greater increments, such as 2mm or greater increments and including 5 mm or greater increments.

Where the imaging sensor is configured to capture images at differentpositions along a path of the flow stream, the imaging sensor may beconfigured to capture images continuously or in discrete intervals. Insome instances, imaging sensors of interest are configured to captureimages continuously. In other instances, imaging sensors are configuredto take measurements in discrete intervals, such as capturing an imageof the flow stream every 0.001 millsecond, every 0.01 millsecond, every0.1 millsecond, every 1 millsecond, every 10 millseconds, every 100millseconds and including every 1000 millseconds, or some otherinterval.

As described in greater detail below, the imaging sensor is configuredto capture one or more images of the flow stream in each detectionfield. For example, the imaging sensor may be configured to capture 2 ormore images of the flow stream in each detection field, such as 3 ormore images, such as 4 or more images, such as 5 or more images, such as10 or more images, such as 15 or more images and including 25 or moreimages. Where a plurality of images are captured in a detection field,the processor (as discussed below) may include digital imagingprocessing algorithm for stitching together the plurality of images.

Depending on the flow stream rate and desired image resolution, theimaging sensor may have an exposure time of 100 ms or less when readingout the full sensor, such as 75 ms or less, such as 50 ms or less, suchas 25 ms or less, such 10 ms or less, such as 5 ms or less, such as 1 msor less, such as 0.1 ms or less such as 0.01 ms or less, such as 0.001ms or less, such as 0.0001 ms or less, such as 0.00001 ms or less andincluding an exposure time of 0.000001 ms or less. For example, theexposure time of the imaging sensor in a detection field which capturesimages of the flow stream at the flow cell nozzle orifice may have anexposure time which ranges from 0.0001 ms to 10 ms, such as from 0.001ms to 5 ms, such as from 0.01 ms to 2 ms and including from 0.1 ms to 1ms. The exposure time of imaging sensors in a detection field whichcaptures images of the flow cytometer flow stream downstream from thenozzle orifice may have an exposure time which ranges from 0.0001 ms to10 ms, such as from 0.001 ms to 5 ms, such as from 0.01 ms to 2 ms andincluding from 0.1 ms to 1 ms.

In certain embodiments, imaging sensors in the subject systems may have1M active pixels or more, such as 1.5M or more, e.g., 2M or more, 2.5Mor more, or 3M or more. In certain aspects, a pixel corresponds to anactual physical dimension of about 0.3 μm. Depending on the detectionfield, in some instances, imaging sensors have a sensor area of 150 mm²or more, such as about 150 mm² to about 175 mm², about 175 mm² to about200 mm², 200 mm² to about 225 mm², about 225 mm² to about 250 mm², about250 mm² to about 300 mm², about 300 mm² to about 400 mm², about 400 mm²to about 500 mm², about 500 mm² to about 750 mm², about 750 mm² to about1000 mm², or about 1000 mm² or more.

The imaging sensor may be positioned at any suitable distance from theflow cytometer flow stream so long as the detection field is capable ofcapturing an image of the flow stream. For example, the imaging sensormay be positioned 0.01 mm or more from the flow stream, such as 0.05 mmor more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm ormore, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm ormore, such as 15 mm or more, such as 25 mm or more and including 50 mmor more from the flow cytometer flow stream.

In some embodiments, the imaging sensor is positioned at an angle withrespect to the flow stream axis. For example, the imaging sensor may bepositioned at an angle with respect to the axis of the flow stream whichranges from 10° to 90°, such as from 15° to 85°, such as from 20° to80°, such as from 25° to 75° and including from 30° to 60°. In certainembodiments, the imaging sensor is positioned at a 90° angle withrespect to the axis of the flow stream.

In some instances, the imaging sensor also includes an opticaladjustment protocol. By “optical adjustment” is meant that capturingimages of the detection field by the imaging sensor may be changed asdesired, such as to increase or decrease the captured dimensions or toenhance the optical resolution of the image. In some instances, opticaladjustment is a magnification protocol configured to increase the sizeof the detection field captured by the imaging sensor, such as by 5% orgreater, such as by 10% or greater, such as by 25% or greater, such asby 50% or greater and including increasing the detection field of theimaging sensor by 75% or greater. In other instances, optical adjustmentis a de-magnification protocol configured to decrease the size of thedetection field captured by the imaging sensor, such as by 5% orgreater, such as by 10% or greater, such as by 25% or greater, such asby 50% or greater and including decreasing the width of the slit shapedbeam by 75% or greater. In certain embodiments, optical adjustment is anenhanced resolution protocol configured to improve the resolution of thecaptured images, such as by 5% or greater, such as by 10% or greater,such as by 25% or greater, such as by 50% or greater and includingenhancing the resolution of the captured images by 75% or greater.Capturing images of the detection field by the imaging sensor may beadjusted with any convenient optical adjustment protocol, including butnot limited to lens, mirrors, filters and combinations thereof. Incertain embodiments, the imaging sensor includes a focusing lens. Thefocusing lens, for example may be a de-magnifying lens. In otherembodiments, the focusing lens is a magnifying lens.

Imaging sensors of the present disclosure may also include one or morewavelength separators. The term “wavelength separator” is used herein inits conventional sense to refer to an optical protocol for separatingpolychromatic light into its component wavelengths for detection.Wavelength separation, according to certain embodiments, may includeselectively passing or blocking specific wavelengths or wavelengthranges of the polychromatic light. To separate wavelengths of light, thetransmitted light may be passed through any convenient wavelengthseparating protocol, including but not limited to colored glass,bandpass filters, interference filters, dichroic mirrors, diffractiongratings, monochromators and combinations thereof, among otherwavelength separating protocols. Depending on the detection field, lightsource and flow stream being visualized, systems may include one or morewavelength separators, such as two or more, such as three or more, suchas four or more, such as five or more and including 10 or morewavelength separators. In one example, imaging sensors include onebandpass filter. In another example, imaging sensors include two or morebandpass filters. In another example, imaging sensors include two ormore bandpass filters and a diffraction grating. In yet another example,imaging sensors include a plurality of bandpass filters and amonochromator. In certain embodiments, imaging sensors include aplurality of bandpass filters and diffraction gratings configured into afilter wheel setup. Where imaging sensors include two or more wavelengthseparators, the wavelength separators may be utilized individually or inseries to separate polychromatic light into component wavelengths. Insome embodiments, wavelength separators are arranged in series. In otherembodiments, wavelength separators are arranged individually such thatone or more measurements are conducted using each of the wavelengthseparators.

In some embodiments, systems include one or more optical filters, suchas one or more bandpass filters. For example, in some instances theoptical filters of interest are bandpass filters having minimumbandwidths ranging from 2 nm to 100 nm, such as from 3 nm to 95 nm, suchas from 5 nm to 95 nm, such as from 10 nm to 90 nm, such as from 12 nmto 85 nm, such as from 15 nm to 80 nm and including bandpass filtershaving minimum bandwidths ranging from 20 nm to 50 nm. In otherinstances, the optical filters are longpass filters, such as for examplelongpass filters which attenuate wavelengths of light of 1600 nm orless, such as 1550 nm or less, such as 1500 nm or less, such as 1450 nmor less, such as 1400 nm or less, such as 1350 nm or less, such as 1300nm or less, such as 1000 nm or less, such as 950 nm or less, such as 900nm or less, such as 850 nm or less, such as 800 nm or less, such as 750nm or less, such as 700 nm or less, such as 650 nm or less, such as 600nm or less, such as 550 nm or less, such as 500 nm or less and includinga longpass filter which attenuates wavelengths of light of 450 nm orless. In yet other instances, the optical filters are shortpass filters,such as for example shortpass filters which attenuate wavelengths oflight of 200 nm or greater, such as 250 nm or greater, such as 300 nm orgreater, such as 350 nm or greater, such as 400 nm or greater, such as450 nm or greater, such as 500 nm or greater, such as 550 nm or greaterand including shortpass filters which attenuate wavelengths of light of600 nm or greater.

In other embodiments, the wavelength separator is a diffraction grating.Diffraction gratings may include, but are not limited to transmission,dispersive or reflective diffraction gratings. Suitable spacings of thediffraction grating may vary depending on the configuration of the lightsource, detection field and imaging sensor and other optical adjustprotocols present (e.g., focusing lens), ranging from 0.01 μm to 10 μm,such as from 0.025 μm to 7.5 μm, such as from 0.5 μm to 5 μm, such asfrom 0.75 μm to 4 μm, such as from 1 μm to 3.5 μm and including from 1.5μm to 3.5 μm.

In some embodiments, each imaging sensor is operably coupled to one ormore light sources for illuminating the flow stream in the detectionfield. In some embodiments, the light source is a broadband lightsource, emitting light having a broad range of wavelengths, such as forexample, spanning 50 nm or more, such as 100 nm or more, such as 150 nmor more, such as 200 nm or more, such as 250 nm or more, such as 300 nmor more, such as 350 nm or more, such as 400 nm or more and includingspanning 500 nm or more. For example, one suitable broadband lightsource emits light having wavelengths from 200 nm to 1500 nm. Anotherexample of a suitable broadband light source includes a light sourcethat emits light having wavelengths from 400 nm to 1000 nm. Anyconvenient broadband light source protocol may be employed, such as ahalogen lamp, deuterium arc lamp, xenon arc lamp, stabilizedfiber-coupled broadband light source, a broadband LED with continuousspectrum, superluminescent emitting diode, semiconductor light emittingdiode, wide spectrum LED white light source, an multi-LED integratedwhite light source, among other broadband light sources or anycombination thereof.

In other embodiments, the light source is a narrow band light sourceemitting a particular wavelength or a narrow range of wavelengths. Insome instances, the narrow band light sources emit light having a narrowrange of wavelengths, such as for example, 50 nm or less, such as 40 nmor less, such as 30 nm or less, such as 25 nm or less, such as 20 nm orless, such as 15 nm or less, such as 10 nm or less, such as 5 nm orless, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Any convenientnarrow band light source protocol may be employed, such as a narrowwavelength LED, laser diode or a broadband light source coupled to oneor more optical bandpass filters, diffraction gratings, monochromatorsor any combination thereof.

The subject systems may include one or more light sources, as desired,such as two or more light sources, such as three or more light sources,such as four or more light sources, such as five or more light sourcesand including ten or more light sources. The light source may include ancombination of types of light sources, for example, where two lightssources are employed, a first light source may be a broadband whitelight source (e.g., broadband white light LED) and second light sourcemay be a broadband near-infrared light source (e.g., broadband near-IRLED). In other instances, where two light sources are employed, a firstlight source may be a broadband white light source (e.g., broadbandwhite light LED) and the second light source may be a narrow spectralight source (e.g., a narrow band visible light or near-IR LED). In yetother instances, the light source is an plurality of narrow band lightsources each emitting specific wavelengths, such as an array of two ormore LEDs, such as an array of three or more LEDs, such as an array offive or more LEDs, including an array of ten or more LEDs.

In some embodiments, light sources emit light having wavelengths rangingfrom 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300nm to 1000 nm, such as from 350 nm to 900 nm and including from 400 nmto 800 nm. For example, the light source may include a broadband lightsource emitting light having wavelengths from 200 nm to 900 nm. In otherinstances, the light source includes a plurality of narrow band lightsources emitting wavelengths ranging from 200 nm to 900 nm. For example,the light source may be plurality of narrow band LEDs (1 nm-25 nm) eachindependently emitting light having a range of wavelengths between 200nm to 900 nm. In some embodiments, the narrow band light source is oneor more narrow band lamps emitting light in the range of 200 nm to 900nm, such as a narrow band cadmium lamp, cesium lamp, helium lamp,mercury lamp, mercury-cadmium lamp, potassium lamp, sodium lamp, neonlamp, zinc lamp or any combination thereof.

In certain embodiments, the light source is a stroboscopic light sourcewhere the flow stream is illuminated with periodic flashes of light.Depending on the light source (e.g., flash lamp, pulsed laser) thefrequency of light strobe may vary, and may be 0.01 kHz or greater, suchas 0.05 kHz or greater, such as 0.1 kHz or greater, such as 0.5 kHz orgreater, such as 1 kHz or greater, such as 2.5 kHz or greater, such as 5kHz or greater, such as 10 kHz or greater, such as 25 kHz or greater,such as 50 kHz or greater and including 100 kHz or greater. In theseembodiments, the strobe light may be operably coupled to a processorhaving a frequency generator which regulates strobe frequency. In someinstances, the frequency generator is coupled to the droplet drivegenerator such that the strobe light is synchronized with dropletgeneration. In other instances, the frequency generator of the strobelight is operably coupled to the one or more optical sensors such thatthe frequency of the strobe light is synchronized with the frequency ofimage capture. In certain instances, suitable strobe light sources andfrequency controllers include, but are not limited to those described inU.S. Pat. Nos. 5,700,692 and 6,372,506, the disclosures of which areherein incorporated by reference. Strobing and pulsed light sources arealso described in Sorenson, et al. Cytometry, Vol. 14, No. 2, pages115-22 (1993); Wheeless, et al. The Journal of Histochemestry andCytochemistry, Vol. 24, No. 1, pages 265-268 (1976), the disclosures ofwhich are herein incorporated by reference.

As summarized above, systems include one or more processors operablycoupled to the imaging sensors where the processors are configured togenerate a data signal from the captured images and to adjust one ormore parameters of the flow cytometer in response to the data signal. Inembodiments, the processor is configured to execute instructions frommemory to adjust one or more parameters of the flow cytometer based onthe data signal derived from the captured images. Parameters of the flowcytometer which may be adjusted according to embodiments of the presentdisclosure include, but are not limited to hydrostatic pressure, dropcharging voltage, deflection plate voltage, charge correction value,drop delay, drop drive frequency, drop amplitude and charge phase. Incertain embodiments, the processor is operably coupled to one or moresupport stages and the positioning of the support stages may be adjustedin response to the data signal derived from the captured images.

In embodiments, the processors include memory having a plurality ofinstructions for performing the steps of the subject methods (asdescribed in greater detail below), such as illuminating a flowcytometer flow stream in a detection field with a light source,capturing one or more images of the flow stream, generating a datasignal corresponding to one or more properties of the flow stream basedon the captured images, and adjusting parameters of the flow cytometerin response to the data signal. The subject systems may include bothhardware and software components, where the hardware components may takethe form of one or more platforms, e.g., in the form of servers, suchthat the functional elements, i.e., those elements of the system thatcarry out specific tasks (such as managing input and output ofinformation, processing information, etc.) of the system may be carriedout by the execution of software applications on and across the one ormore computer platforms represented of the system. The processorincludes memory having instructions stored thereon for performing thesteps of the subject methods including illuminating a flow cytometerflow stream in a detection field with a light source, capturing one ormore images of the flow stream, generating a data signal correspondingto one or more properties of the flow stream based on the capturedimages, and adjusting parameters of the flow cytometer in response tothe data signal.

In embodiments, the processor is configured to generate a data signalcorresponding to one or more properties of the flow stream from thecaptured images. In detection fields where the flow stream iscontinuous, the processor may be configured to generate a data signalcorresponding to the spatial position of the flow stream, the dimensionsof the flow stream such as flow stream width, as well as flow rate andflow turbulence. In detection fields where the flow stream is composedof discrete droplets, the processor may be configured to generate a datasignal corresponding to the spatial position of the flow stream, dropsize including drop diameter and volume, drop drive frequency, dropamplitude as well as the uniformity of drop size and frequency. Incertain embodiments, the processor may be configured to generate a datasignal corresponding to the ratio of the size of the flow stream ascompared to the expected size of the flow stream based on empiricalcharacteristics of the flow cytometer and user inputted data. In otherembodiments, the processor may be configured to assess the capturedimages to determine whether a flow stream is present or absent in aparticular detection field. In yet other embodiments, the processor maybe configured to assess the captured images of the flow stream todetermine the flow cell nozzle orifice size.

In some embodiments, the processor is operably coupled to an imagingsensor which captures images of the flow stream in a detection field andgenerates a data signal corresponding to the spatial position of theflow stream. For instance, the processor may take the captured images ofthe flow stream in the detection field and map the spatial position ofthe flow stream in an X-Y plane. In some instances, the position of theflow stream in the X-Y plane is compared to the spatial position of thevertical axis of the flow cell nozzle to determine position of the flowstream with respect to the vertical axis formed by the flow cell nozzle.Based on the determined spatial position of the flow stream in thedetection field, the processor generates a data signal corresponding tothe spatial position of the flow stream.

In these embodiments, the data signal corresponding to the spatialposition of the flow stream may be used by the processor toautomatically adjust one or more parameters of the flow cytometer. Insome instances, the data signal is used to adjust the position of asupport stage having one or more containers for collecting particles,such as for cell sorting. In certain embodiments, the processorgenerates a data signal corresponding to the position of the flow streamand adjusts the position of a support stage so that the collectioncontainers on the support stage are aligned with the trajectory of theflow stream. For example, the processor may be configured to map theposition of the flow stream in each detection field in an X-Y plane, mapthe position of the container in the X-Y plane and match the position ofthe container in the X-Y plane with the position of the flow stream inthe X-Y plane to align the collection container with the flow stream. Insome instances, the subject systems are configured to map the positionof the flow stream in two detection fields. In these instances, theprocessor maps the spatial position of the flow stream in the firstdetection field in an X-Y plane and maps the spatial position of theflow stream in the second detection field in the X-Y plane. Based on themapped positions of the flow stream in the first and second detectionfields, the processor is configured to generate a data signalcorresponding to the spatial position of the flow stream in the flowcytometer.

In these embodiments, the processor generates a data signalcorresponding to the spatial position of the flow stream andautomatically adjusts the position of a support stage in an X-Y plane soas to optimize collection of the flow stream. For example, optimizingcollection may include reducing the number of flow stream particles notcollected by the containers on the support stage due to misalignment ofthe flow stream with the collection containers. For example, the numberof particles not collected by containers on the support stage due tomisalignment is reduced by 5% or more as compared to a container on asupport stage not adjusted in response to the data signal, such as by10% or more, such as 15% or more, such as 20% or more, such as 25% ormore, such as 35% or more, such as 50% or more, such as 75% or more,such as 90% or more, such as 95% or more and including by 99% or more.Put another way, the processer in certain instances automatically alignsthe position of the support stage in response to data signalcorresponding to the spatial position of the flow stream so that thenumber of particles collected by the container is increased by 5% ormore as compared to a container on a support stage not adjusted inresponse to the data signal, such as by 10% or more, such as 15% ormore, such as 20% or more, such as 25% or more, such as 35% or more,such as 50% or more, such as 75% or more, such as 90% or more, such as95% or more and including by 99% or more. In other instances, adjustingthe position of the support stage having containers for collectingcharged particles during cell sorting may be increased as compared tocollection with a support stage not adjusted in response to the datasignal by 2 fold or greater, such as 3 fold or greater, such as 5 foldor greater and including by 10 fold or greater.

In some embodiments, a support stage is positioned downstream fromdeflector plates and includes containers for collecting sorted cellsthat have been separated based on charge (i.e., positive, negative andneutral). In some instances, the support structure may include three ormore containers. In other instances, the support structure includes asingle container partitioned into three or more compartments forcollecting the sorted cells. An imaging sensor is configured to captureimages of the flow stream in a detection field downstream from thedeflector plates and a processor operably coupled to the imaging sensorgenerates a data signal corresponding to the spatial positions of theflow streams. In these embodiments, the processor takes the capturedimages of each flow stream and maps the spatial position of the flowstream in an X-Y plane. In some instances, the position of the flowstream in the X-Y plane is compared to the position of the flow streambefore entering the deflector plates to determine the deviation due tothe effects of the deflector plates. In these embodiments, the processormay generate a distinct data signal corresponding to the position of theflow stream of neutral particles, the flow stream of negative particlesand the flow stream of positive particles, or any combination thereof.In one example, the processor generates a data signal which correspondsto the flow stream position of neutral particles after deflection by thedeflector plates. In another example, the processor generates a datasignal which corresponds to the flow stream position of negativeparticles after deflection by the deflector plates. In yet anotherexample, the processor generates a data signal which corresponds to theflow stream position of positive particles coming from the deflectorplates. In still another example, the processor generates a data signalwhich corresponds to the flow stream positions of the positiveparticles, the negative particles and the neutral particles.

Based on the determined spatial positions of each flow stream, theprocessor automatically adjusts one or more parameters of the flowcytometer. For instance, the data signal may be used to adjust theposition of a support stage having containers for collecting thepositive particles, the negative particles and the neutral particles. Inthese instances, the processor generates a data signal corresponding tothe spatial positions of each flow stream (i.e., neutral particlestream, positive particle stream and negative particle stream) andautomatically adjusts the position of the support stage to aligncollection containers with each of the flow streams so as to optimizecollection. For example, the position of the support stage may beautomatically adjusted to align collection containers with each flowstream so that the number of particles collected by the containers isincreased by 5% or more as compared to a container on a supportstructure not adjusted in response to the data signal, such as by 10% ormore, such as 15% or more, such as 20% or more, such as 25% or more,such as 35% or more, such as 50% or more, such as 75% or more, such as90% or more, such as 95% or more and including by 99% or more.

As summarized above, systems according to embodiments of the presentdisclosure include one or more processors that are automated to adjustparameters of a flow cytometer based on data signals derived fromcaptured images of the flow cytometer flow stream. In certainembodiments, parameters of the flow cytometer which may be adjustedinclude sheath fluid pressure, hydrostatic pressure, droplet chargingvoltage, deflection plate voltage, charge correction value, drop delay,drop drive frequency, drop amplitude and charge phase.

In some embodiments, the processor may be configured to adjust thehydrostatic pressure in response to a data signal corresponding to oneor more properties of the flow stream determined based on the capturedimages. In some instances, the hydrostatic pressure may be increasedsuch as by 0.1 psi or more, such as 0.5 psi or more, such as by 1 psi ormore, such as by 5 psi or more, such as by 10 psi or more, such as by 25psi or more, such as by 50 psi or more, such as by 75 psi or more andincluding increasing the hydrostatic pressure by 100 psi or more. Forexample, the hydrostatic pressure may be increased by 1% or more, suchas by 5% or more, such as by 10% or more, such as by 15% or more, suchas by 25% or more, such as by 50% or more, such as by 75% or more andincluding by increasing the hydrostatic pressure by 90% or more. Inother instances, the hydrostatic pressure is reduced, such as by 0.1 psior more, such as 0.5 psi or more, such as by 1 psi or more, such as by 5psi or more, such as by 10 psi or more, such as by 25 psi or more, suchas by 50 psi or more, such as by 75 psi or more and including reducingthe hydrostatic pressure by 100 psi or more. For example, thehydrostatic pressure may be reduced by 1% or more, such as by 5% ormore, such as by 10% or more, such as by 15% or more, such as by 25% ormore, such as by 50% or more, such as by 75% or more and includingreducing the hydrostatic pressure by 90% or more.

In yet other embodiments, the processor may be configured to adjust thedrop charging voltage in response to a data signal corresponding to oneor more properties of the flow stream determined based on the capturedimages. In some instances, the drop charging voltage is increased, suchas by 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more,such as by 0.5V or more, such as by 1V or more, such as by 5V or more,such as by 10V or more, such as by 15V or more, such as by 25V or more,such as by 50V or more and including increasing the drop chargingvoltage by 75V or more. For example, the drop charging voltage may beincreased by 1% or more, such as by 5% or more, such as by 10% or more,such as by 15% or more, such as by 25% or more, such as by 50% or more,such as by 75% or more and including increasing the drop chargingvoltage by 90% or more. In other instances, the drop charging voltage isreduced, such as by 0.01 V or more, such as 0.05 V or more, such as 0.1V or more, such as by 0.5V or more, such as by 1V or more, such as by 5Vor more, such as by 10V or more, such as by 15V or more, such as by 25Vor more, such as by 50V or more and including reducing the drop chargingvoltage by 75V or more. For example, the drop charging voltage may bereduced by 1% or more, such as by 5% or more, such as by 10% or more,such as by 15% or more, such as by 25% or more, such as by 50% or more,such as by 75% or more and including reducing the drop charging voltageby 90% or more.

In yet other embodiments, the processor may be configured to adjust thedeflection plate voltage in response to a data signal corresponding toone or more properties of the flow stream determined based on thecaptured images. In some instances, the deflection plate voltage isincreased, such as by 5V or more, such as by 10V or more, such as by 50Vor more, such as by 100V or more, such as by 250V or more, such as by500V or more, such as by 1000V or more and including increasing the dropcharging voltage by 2000V or more. For example, the deflection platevoltage may be increased by 1% or more, such as by 5% or more, such asby 10% or more, such as by 15% or more, such as by 25% or more, such asby 50% or more, such as by 75% or more and including increasing thedeflection plate voltage by 90% or more. In other instances, the dropcharging voltage is reduced, such as by 0.5V or more, such as by 5V ormore, such as by 10V or more, such as by 50V or more, such as by 100V ormore, such as by 250V or more, such as by 500V or more, such as by 1000Vor more and including reducing the deflection plate voltage by 2000V ormore. For example, the deflection plate voltage may be reduced by 1% ormore, such as by 5% or more, such as by 10% or more, such as by 15% ormore, such as by 25% or more, such as by 50% or more, such as by 75% ormore and including reducing the deflection plate voltage by 90% or more.

In still other embodiments, the processor may be configured to adjustthe drop drive frequency in response to a data signal corresponding toone or more properties of the flow stream determined based on thecaptured images. In some instances, the drop drive frequency isincreased, such as by 0.01 Hz or more, such as by 0.05 Hz or more, suchas by 0.1 Hz or more, such as by 0.25 Hz or more, such as by 0.5 Hz ormore, such as by 1 Hz or more, such as by 2.5 Hz or more, such as by 5Hz or more, such as by 10 Hz or more and including by 25 Hz or more. Forexample, the drop drive frequency may be increased by 1% or more, suchas by 5% or more, such as by 10% or more, such as by 15% or more, suchas by 25% or more, such as by 50% or more, such as by 75% or more andincluding increasing the drop drive frequency by 90% or more. In otherinstances, the drop drive frequency is reduced, such as by 0.01 Hz ormore, such as by 0.05 Hz or more, such as by 0.1 Hz or more, such as by0.25 Hz or more, such as by 0.5 Hz or more, such as by 1 Hz or more,such as by 2.5 Hz or more, such as by 5 Hz or more, such as by 10 Hz ormore and including by 25 Hz or more. For example, the drop drivefrequency may be reduced by 1% or more, such as by 5% or more, such asby 10% or more, such as by 15% or more, such as by 25% or more, such asby 50% or more, such as by 75% or more and including reducing the dropfrequency by 90% or more. In still other embodiments, the processor maybe configured to adjust the drop delay in response to a data signalcorresponding to one or more properties of the flow stream determinedbased on the captured images. In some instances, the drop delay isincreased, such as by 0.01 microseconds or more, such as by 0.05microseconds or more, such as by 0.1 microseconds or more, such as by0.3 microseconds or more, such as by 0.5 microseconds or more, such asby 1 microseconds or more, such as by 2.5 microseconds or more, such asby 5 microseconds or more, such as by 7.5 microseconds or more andincluding increasing the drop delay by 10 microseconds or more. Forexample, the drop delay may be increased by 1% or more, such as by 5% ormore, such as by 10% or more, such as by 15% or more, such as by 25% ormore, such as by 50% or more, such as by 75% or more and includingincreasing the drop delay by 90% or more. In other instances, the dropfrequency is reduced, such as by 0.01 microseconds or more, such as by0.05 microseconds or more, such as by 0.1 microseconds or more, such asby 0.3 microseconds or more, such as by 0.5 microseconds or more, suchas by 1 microseconds or more, such as by 2.5 microseconds or more, suchas by 5 microseconds or more, such as by 7.5 microseconds or more andincluding reducing the drop delay by 10 microseconds or more. Forexample, the drop delay may be reduced by 1% or more, such as by 5% ormore, such as by 10% or more, such as by 15% or more, such as by 25% ormore, such as by 50% or more, such as by 75% or more and includingreducing the drop delay by 90% or more.

In still other embodiments, the processor may be configured to adjustthe drop amplitude in response to a data signal corresponding to one ormore properties of the flow stream determined based on the capturedimages. In some instances, the drop amplitude is increased, such as by0.01 volts or more, such as by 0.025 volts or more, such as by 0.05volts or more, such as by 0.1 volts or more, such as by 0.25 volts ormore, such as by 0.5 volts or more and including increasing the dropamplitude by 1 volt or more. For example, the drop amplitude may beincreased by 1% or more, such as by 5% or more, such as by 10% or more,such as by 15% or more, such as by 25% or more, such as by 50% or more,such as by 75% or more and including increasing the drop amplitude by90% or more. In other instances, the drop amplitude is reduced, such asby 0.01 volts or more, such as by 0.025 volts or more, such as by 0.05volts or more, such as by 0.075 volts or more, such as by 0.1 volts ormore, such as by 0.25 volts or more and including reducing the dropamplitude by 1 volt or more. For example, the drop amplitude may bereduced by 1% or more, such as by 5% or more, such as by 10% or more,such as by 15% or more, such as by 25% or more, such as by 50% or more,such as by 75% or more and including reducing the drop amplitude by 90%or more.

In some embodiments, the processor is operably coupled to an imagingsensor which captures images of a flow cytometer flow stream in adetection field and generates a data signal corresponding to thephysical dimensions of the flow stream based on the captured images.Where the flow stream is a continuous stream, in some instances theprocessor is configured to take the captured images and generate a datasignal corresponding to the width of the flow stream. In detectionfields where the flow stream is composed of discrete droplets, in someinstances the processor is configured to generate a data signalcorresponding to droplet diameter.

In certain embodiments, the processor may be configured to compare thephysical dimensions of the flow stream determined from the capturedimages with dimensions expected based on empirical characteristics ofthe flow cytometer (such as flow cell nozzle orifice size and sheathfluid pressure) and inputted parameters by the user. In theseembodiments, the processor is configured to generate a data signalcorresponding to the ratio of the physical dimensions of the flow streamas compared to the expected flow stream dimensions based on theempirical characteristics of the flow cytometer and user inputtedparameters. For example, the processor may be configured to generate adata signal which indicates that the flow stream is 99% or less of theexpected size of the flow stream based on the empirical characteristicsof the flow cytometer and user inputted parameters, such as 95% or less,such as 90% or less, such as 85% or less, such as 80% or less, such as75% or less, such as 50% or less, such as 25% or less and including 10%or less of the expected size of the flow stream. In other embodiments,the processor may be configured to generate a data signal whichindicates that the flow stream is greater than the size expected basedon the empirical characteristics of the flow cytometer and user inputtedparameters, such as being 105% or greater of the size of the flowstream, such a 110% or greater, such as 125% or greater and including150% or greater. In these embodiments, the processor may be configuredto automate adjustments to one or more parameters of the flow cytometerbased on the data signal corresponding to the ratio of the flow streamsize from the captured images and the expected size of the flow streambased on empirical characteristics of the flow cytometer and user input.For example, the processor may be configured to automatically adjust thepump rate, the hydrostatic pressure and drop drive frequency in responseto the determined ratio.

In certain instances, the processor is configured for determining a flowcell nozzle opening diameter. In these embodiments, the processor isoperably coupled to an imaging sensor which captures images of the flowstream at the orifice of the flow cell nozzle and generates a datasignal corresponding to the physical dimensions of the flow stream.Based on the data signal corresponding to the physical dimensions of theflow stream, the processor is configured to determine the flow cellnozzle opening diameter. In some instances, based on the data signalcorresponding to the physical dimensions of the flow stream theprocessor may determine that the flow cell nozzle opening diameter is 25μm or greater, such as 35 μm or greater, such as 45 μm or greater, suchas 50 μm or greater, such as 60 μm or greater, such as 75 μm or greater,such as 100 μm or greater and including 150 μm or greater. For example,the system may be configured to determine a flow cell nozzle openingdiameter from the physical dimensions of the flow stream that rangesfrom 25 μm to 200 μm, such as from 35 μm to 175 μm, such as from 50 μmto 150 μm and including from 75 μm to 100 μm.

In certain instances, the nozzle opening diameter is determined based onthe width of the flow stream. In other instances, the nozzle openingdiameter is determined based on droplet volume.

The processor may, in certain instances, be configured to automaticallyadjust one or more parameters based on the determined nozzle openingdiameter, such as for example, the hydrostatic pressure, the sheathfluid pressure, drop charge, deflection voltage, charge correctionvalue, drop delay, drop drive frequency, drop amplitude charge phase andany combinations thereof, as discussed above.

In some embodiments, the processor may be configured to automaticallyadjust the drop drive frequency in response to the data signalcorresponding to the flow cell nozzle orifice size determined using thecaptured images of the flow stream. For example, the drop drivefrequency may be increased by 0.01 Hz or more, such as by 0.05 Hz ormore, such as by 0.1 Hz or more, such as by 0.25 Hz or more, such as by0.5 Hz or more, such as by 1 Hz or more, such as by 2.5 Hz or more, suchas by 5 Hz or more, such as by 10 Hz or more and including by 25 Hz ormore. In other instances, the processor is configured to automaticallyreduce the drop drive frequency in response to the flow cell nozzleorifice size determined using the captured images of the flow stream,such as by 0.01 Hz or more, such as by 0.05 Hz or more, such as by 0.1Hz or more, such as by 0.25 Hz or more, such as by 0.5 Hz or more, suchas by 1 Hz or more, such as by 2.5 Hz or more, such as by 5 Hz or more,such as by 10 Hz or more and including by 25 Hz or more.

In other embodiments, the processor may be configured to automaticallyadjust the sheath fluid pressure in response to the data signalcorresponding to the flow cell nozzle orifice size determined using thecaptured images of the flow stream. For example, the sheath fluidpressure may be increased by 0.001 psi or more, such as 0.005 psi ormore, such as by 0.01 psi or more, such as by 0.05 psi or more, such asby 0.1 psi or more, such as 0.5 psi or more, such as by 1 psi or more,such as by 5 psi or more, such as by 10 psi or more, such as by 25 psior more, such as by 50 psi or more, such as by 75 psi or more andincluding increasing the sheath fluid pressure by 100 psi or more. Inother instances, the processor is configured to automatically reduce thesheath fluid pressure in response to the flow cell nozzle orifice sizedetermined using the captured images of the flow stream, such as by 0.1psi or more, such as 0.5 psi or more, such as by 1 psi or more, such asby 5 psi or more, such as by 10 psi or more, such as by 25 psi or more,such as by 50 psi or more, such as by 75 psi or more and includingreducing the sheath fluid pressure by 100 psi or more.

In some embodiments, systems of interest include an imaging sensorconfigured to capture images in a detection field at the break-off pointof the flow stream. The term “break-off point” is used herein in itsconventional sense to refer to the point at which the continuous flowstream begins to form droplets. In these embodiments, the subjectsystems include a processor operably coupled to the imaging sensor andconfigured to generate a data signal corresponding to the drop volume ofdroplets downstream from the break-off point. The processor takes thecaptured images of the flow stream droplets and measures the dropvolume. The data signal corresponding to the drop volume may be used bythe processor to automatically adjust one or more parameters of the flowcytometer.

In some embodiments, the data signal corresponding to drop volume isused by the processor to automatically adjust the drop drive frequencyof the flow stream. For example, the processor may be configured toautomatically reduce the drop drive frequency, such as by 5% or more,such as by 10% or more, such as by 15% or more, such as by 25% or more,such as by 50% or more, such as by 75% or more, such as by 90% or more,such as by 95% or more and including by 99% or more. In other instances,the processor is configured to automatically reduce the drop drivefrequency by 2-fold or more in response to the data signal correspondingto the determined drop volume, such as by 3-fold or more, such as by4-fold or more, such as by 5-fold or more and including by 10-fold ormore. In yet other instances, the processor is configured toautomatically increase the drop drive frequency, such as by 5% or morein response to the data signal corresponding to drop volume, such as by10% or more, such as by 15% or more, such as by 25% or more, such as by50% or more, such as by 75% or more, such as by 90% or more, such as by95% or more and including by 99% or more. In still other instances, theprocessor is configured to automatically increase the drop drivefrequency by 2-fold or more, such as by 3-fold or more, such as by4-fold or more, such as by 5-fold or more and including by 10-fold ormore.

In other embodiments, the data signal corresponding to the drop volumeis used by the processor to automate sample collection volume duringcell sorting. For example, the volume desired for each collected samplemay be input into the processor and based on the data signalcorresponding to the drop volume, the flow cytometer may be automated tostop collection of the sample after a predetermined amount of time, suchas by removing the collection container or by ceasing the flow stream bythe flow cytometer.

In some embodiments, the processor may be configured to determine thepresence or absence of a flow stream in a detection field. Systems ofinterest may include an imaging sensor configured to capture images ofthe flow cytometer flow stream exiting the orifice of the flow cellnozzle and a processor operably coupled to the imaging sensor configuredto assess the captured images to determine whether a flow stream ispresent or not present in the detection field. For example, determiningwhether a flow stream is present or not present in captured images ofthe flow cell nozzle orifice may be used to determine whether the flowcell has a clogged nozzle. In these embodiments, captured images by theimaging sensors are assessed by the processor and if a flow stream isdetected in the images by the processor, the processor is configured togenerate a signal indicating the presence of a flow stream. On the otherhand, if after assessing the captured images, the processor determinesthat the flow stream is absent in the captured images, the processor maybe configured to generate a signal indicating the absence of a flowstream.

Where the processor determines that no flow stream is present in thecaptured images, in certain embodiments, the subject systems areconfigured to automatically alert a user that the absence of flow streamis a result of flow cytometer malfunction, such as a clogged nozzle. Inthese embodiments, the processor correlates the data signalcorresponding to the absence of a flow stream with input from the useras to whether a flow stream should be expected. In some embodiments, auser may configure the system to have a “closed loop” configurationwhere flow stream from the nozzle is directed to a waste receptaclewithout forming a flow stream. In these embodiments, the flow cytometerdoes not alert the user of a malfunction (e.g., clogged nozzle) since aflow stream is not expected. However, where a flow stream is expected(such as during normal use), the processor is automated to alert theuser of a malfunction if after assessing the captured images no flowstream is detected.

In certain embodiments, after the processor has generated a data signalcorresponding to one or more properties of the flow stream based on thecaptured images, an output module may communicate the parameters of theflow cytometer may be adjusted in response to the data signal. In someinstances, the output module communicates an output in conjunction withthe subject systems adjusting parameters of the flow cytometer. In otherinstances, the output module communicates the parameters beforeadjustment and may require confirmation of adjustment by the user.Output from the processor may be communicated to the user by anyconvenient protocol, such as for example by displaying on a monitor orby printing a report.

As discussed above, systems in some embodiments include one or moresupport stages operably coupled to the processors. Suitable supportstages may be any convenient mounting device configured to hold in placeone or more components of the subject systems, such as planar substrate,contoured mounting devices, cylindrical or tubular support structures,laser or LED holders, among other types of support structures. In someinstances, the support stage is a mount for an illumination device, suchas a laser or an LED. In other instances, systems include a supportstructure for holding one or more containers for collecting particlesfrom the flow stream. For example, the support stage may be configuredto hold in place containers including, but are not limited to testtubes, conical tubes, multi-compartment containers such as microtiterplates (e.g., 96-well plates), centrifuge tubes, culture tubes,microtubes, caps, cuvettes, bottles, rectilinear polymeric containers,among other types of containers.

Systems of interest may include one or more support stages, as desired,such as two or more, such as three or more, such as four or more andincluding five or more support stages. For example, the number ofsupport stages may range from 1 to 10 support stages, such as from 2 to7 support stages and including from 3 to 5 support stages. In certainembodiments, systems of interest include one support stage. In otherembodiments, systems include two support stages. In one example, thesubject systems include a support stage having a container forcollecting droplets from the flow stream. In another example, thesubject systems include a support stage having a mounted laser. In yetanother example, the subject system includes a first support stagehaving a mounted laser and a second support stage having a container forcollecting droplets from the flow stream.

In some embodiments, support stages are movable. For instance, in oneexample the support stage may be moved to adjust the position collectioncontainers on the support stage so that they are aligned with the flowstream. In another example, the support stage may be moved to adjust theposition of a laser. In some instances, the support stage is moved intwo dimensions, such as in an X-Y plane orthogonal to the axis of theflow stream. In other instances, the support structure is moved in threedimensions. Where the support stage is configured to move, the supportstage may be moved continuously or in discrete intervals. In someembodiments, the support stage is moved in a continuous motion. In otherembodiments, the support stage is moved in discrete intervals, such asfor example in 0.01 micron or greater increments, such as 0.05 micron orgreater, such as 0.1 micron or greater, such as 0.5 micron or greater,such as 1 micron or greater, such as 10 micron or greater, such as 100microns or greater, such as 500 microns or greater, such as 1 mm orgreater, such as 5 mm or greater, such as 10 mm or greater and including25 mm or greater increments.

Any displacement protocol may be employed to move the supportstructures, such as moving the support stages with a motor actuatedtranslation stage, leadscrew translation assembly, geared translationdevice, such as those employing a stepper motor, servo motor, brushlesselectric motor, brushed DC motor, micro-step drive motor, highresolution stepper motor, among other types of motors.

Certain embodiments of the present disclosure may be described withreference to FIG. 1. A flow cytometer 100 employing an embodiment of thepresent invention is illustrated in FIG. 1. As discussed above, the flowcytometer 100 includes flow cell 104, a sample reservoir 106 forproviding a fluid sample, (e.g., blood sample), to the flow cell and asheath reservoir 108 for providing a sheath fluid to the flow cell. Flowcytometer 100 is configured to transport fluid sample having cells in aflow stream to flow cell 104 in conjunction with a laminating flow ofsheath fluid. Analysis of the flow stream at an interrogation zone 103within flow cell 104 may be used to determine properties of a sample andcontrol the sorting parameters (as described herein). Sampleinterrogation protocols may include a source of light (e.g., laser) 112for illuminating the flow stream and one or more detectors 109 (e.g.,photomultiplier tubes (PMTs), charged coupled device (CCD)) or any othersuitable type of light detecting device. Where light from the lightsource intersects the sample stream in interrogation zone 103, the laserlight is scattered by the sample stream fluid and, in particular, by anycells present in the sample stream. A first portion of the scatteredlaser light will propagate in the direction prior to intersecting thesample stream (referred to herein as the forward scatter light). Asecond portion of the laser light intersecting the interrogation pointwill be scattered at an angle different from the direction ofpropagation (referred to herein as side scatter light). Within the flowcell 104, the sheath fluid surrounds the cell stream, and the combinedsheath fluid and cell stream exits the flow cell 104 through a nozzle102 having orifice 110 as flow stream 111. The flow stream may becontinuous flow of fluid or a series of droplets depending on the actionof a droplet generator.

The flow stream 111 exits the nozzle 102 at the nozzle orifice 110 whichmay have any diameter for example, 50 μm, 70 μm, 100 μm, or any othersuitable diameter. The nozzle diameter will affect the properties of aflow stream, such as the stream dimensions, droplet break-off point anddrop volume. To view the flow stream 111, a light source 112, such as anLED strobe, laser or any other illumination device, may optionallyutilized and be positioned in the region of the sample fluid stream 111.A camera 113 or other image collection device may be positioned tocapture an image of the flow stream in a first detection field. In someembodiments the flow stream may comprise a continuous stream or a seriesof droplets. If the flow stream is a continuous flow of liquid, theimage captured by the camera in the detection field may provide a useror controller with sufficient information to determine the positionand/or dimensions of the flow stream.

In some aspects of this invention the camera 113 or other detectiondevice may affect some action in the flow cytometer 100 based on theimage collected by the camera 113. A set-up controller 114 comprising acomputer algorithm may receive the image of the flow stream anddetermine some action to be performed by the flow cytometer,advantageously freeing the user from manual set-up tasks. In someembodiments the diameter of the nozzle opening 110 may be determinedbased on an image analysis of the dimensions of the flow stream 111captured by the camera 113. In some embodiments a set-up controller 114may be operationally connected to the flow cytometer 100 andautomatically initiate the adjustment of a series of parameters in theflow cytometer based upon the nozzle diameter determined from the imagereceived by the camera. The parameters may include any flow cytometricparameter such as hydrostatic pressure, drop charge, deflection voltage,charge correction value, drop delay, drop frequency, drop amplitude, andcharge phase.

The set-up controller 114 may be operationally connected to a fluidicsystem 115 that may control the rate of the flow stream 111 in the flowcytometer 100. The set-up controller 114 may initiate a pause in theflow stream based on an image received from the camera 113.

The image collected from the camera 113 of the flow stream 111 in thedetection field may provide additional information about the position ofthe flow stream in an XY plane. The camera may be operationallyconnected to one or more stages 116, 119 and the position of the stageor stages may be moved in response to a signal from the camera or set-upcontroller connected to the camera. A collection device or lightemitting device such as a laser 117 may be fixed to a stage and bebeneficially aligned to intercept the flow stream 111 in response to theimage from the camera 113. The light emitting device may be aligned tomaximize the amount of light received by the flow stream. A collectiondevice 118 may fixed to the first stage or to a second stage 119 and bealigned to maximize the collection of a flow stream or orient the flowstream with respect to a ‘home position’ on the collection device. Theimproved automatic alignment of the laser or collection device with theflow stream beneficially reduces the manual adjustment of the stage bythe user.

A second camera 120 or data collection device may be positioned belowthe first camera 113 or data collection device and configured to collectan image in a second detection field. The second camera may bepositioned orthogonally in an XY plane relative to the first camera, oroptionally a series of optics may be positioned in an XY plane such thatthe first and second detection fields are orthogonally oriented. Thesecond camera 120 may also be operationally connected to one or moremoveable stages 116, 119 either directly or via the set-up controller114. Collection or analysis devices may be fixed to the stages. Thesecond detection field may be orthogonally oriented relative to thefirst detection field. Images from the second camera may be used torefine the flow stream position determined from the first camera andprovide for improved positioning of one or more stages associated withthe flow stream. Although cameras 113 and 120 are shown as individualdetectors for exemplary purposes, a plurality of cameras can be used todetect the flow stream in a plurality of detection fields. An additionallight source 123 may be utilized to provide sufficient illumination tocapture the image of the flow stream at this position. Alternatively thelaser 117 may provide sufficient illumination. Furthermore, filters orother optics 121 and 122 may be positioned in front of the lightreceiving areas of cameras 113 and 120, respectively, to filter out anylight or to adjust the resolution or direction of the detection field.

The images from the first and/or second camera 113, 120 may be analyzedby a set-up controller 114 to determine any number of properties of theflow stream, such as position of the flow stream in a detection field ordimensions of the flow stream. In some embodiments a signalcorresponding to the location of the flow stream in the detection fieldmay be transmitted to a set-up controller 114 or directly to a movablestage 116, 119 and initiate the automatic alignment of devices orvessels fixed to the stage with respect to the flow stream.

The flow stream may be a series of droplets that are partially deflectedby a pair of deflection plates 124 and become a plurality of streams125, 126, 127. As further illustrated, the flow cytometer may include aplurality of collection vessels 118, 128 and 129 to collect theplurality of flow streams. The collection vessels may be a single vesselwith a multiple wells such as a 96 or 364 well plate or a series ofvessels. In the example shown in FIG. 1, droplets 127 that have beennegatively charged in the interrogation zone will be directed by thepotentials applied to the deflection plates 124 toward collection vessel128. Droplets 126 which have been neither positively nor negativelycharged will not be deflected by the potentials applied to deflectionplates 124, and therefore continue along their original path intocentral collection vessel 118. Droplets 125 which have been positivelycharged will be deflected by the potentials applied to deflection plates124 toward collection vessel 129. Alignment of the collection vesselswith respect to deflected flow streams is essential to maximizingcollections of sorted cells.

The collection vessel or vessels may be automatically aligned bycollecting data from the first and or second camera 113, 120 todetermine the position of the flow streams in an XY plane. Thecollection vessel(s) may be fixed to a movable stage 119 incommunication with the controller 114 or directly with the first orsecond camera 113, 120. The cameras may determine the position of thestream in the detection field, and generate a signal to the controller.The controller 114 may automatically control the position of acollection vessel 118 disposed beneath the flow stream in order tooptimize the position of the collection vessel with respect to the flowstream. In some embodiments the controller may also control themagnitude of the electric charge received by a portion of the droplets.The magnitude of the electric charge may affect the degree of deflectionexperienced by the droplets and hence the position of the droplets inthe XY plane.

The set-up controller 118 may take further action depending parametersinput in the device. One aspect of the invention is the application ofan input value for drop-volume into the set-up controller 114. The dropvolume may be determined by any means such as empirical measurements ofa volume after a set number of drops from a particular nozzle diameterhave been collected in a defined period of time. The drop volume maythen be input into the set-up controller 114. In some embodiments thecontroller may cause the fluidics system 115 to pause after a set volumeis dispensed to a collection vessel 118. This method beneficiallyimproves a collection protocol because the use of a calibrated dropvolume may provide a more accurate determination of collection volumethan conventional methods which rely on drop count to control collectiontimes. Using methods of this invention and the sorted fluid volumeinformation available, an additional “stopping rule” for the sortingprocess may be implemented.

In some aspects of this invention, the set-up controller may be used todistinguish between a clogged nozzle and a closed loop nozzlespecifically designed not to generate a flow stream. The “closed loop”nozzle has an output that is connected to a tubing system that goesdirectly to waste. It does not create an open, sortable stream, and isused for analysis only. It is important to be able to discern thisnozzle from a clogged sorting nozzle that should create an open,sortable stream but is unable for whatever reason. In some embodimentsthe set-up controller electrically senses when the closed loop nozzle isinstalled. The electrical sensing may take any form such as an insertedclosed loop nozzle providing a ground to a ‘pull up’ resistor circuit.If the closed loop nozzle is sensed in this way, an image of a stream isnot expected by the camera, so a clogged nozzle is not erroneouslyreported when a stream image is not seen. For sorting nozzles, a streamimage is expected, and using the area value of that image, a nozzle sizeis determined the appropriate instrument setting values for the nozzleare executed. If a stream image is not seen and an electrical signalsignifying the presence of a closed loop nozzle is not detected, it isdetermined a sorting nozzle is installed and clogged. The set-upcontroller may initiate a series of actions in this event. For example,the user may be notified of a clogged nozzle, the fluidic system may bepaused, or any other action may be initiated.

Methods for Adjusting Parameters of a Flow Cytometer

Aspects of the disclosure also include methods for adjusting one or moreparameters of a flow cytometer. Methods according to certain embodimentsinclude capturing one or more images of a flow stream of the flowcytometer in a detection field, determining one or more properties ofthe flow stream in the detection field, generating a data signalcorresponding to the one or more properties of the flow stream andadjusting one or more parameters of the flow cytometer in response tothe data signal.

As discussed above, the term “adjusting” refers to changing one or morefunctional parameters of the flow cytometer. The desired adjustment mayvary in terms of goal, where in some instances the desired adjustmentsare adjustments that ultimately result in enhanced efficiency of somedesirable parameter, e.g., improved cell sorting accuracy, enhancedparticle collection, identifying component malfunction (e.g., cloggedflow cell nozzle), energy consumption, particle charging efficiency,more accurate particle charging, enhanced particle deflection duringcell sorting, among other adjustments. In embodiments, the subjectmethods reduce or entirely eliminate the need for user input or manualadjustment during sample analysis with a flow cytometer. In certainembodiments, methods of interest may be fully automated, such thatadjustments made in response to data signals corresponding to one ormore parameters of the flow stream require little to no humanintervention or manual input by the user. In certain embodiments,methods include adjusting one or more parameters of the flow cytometerbased on the data signals corresponding to one or more parameters of theflow stream without any human intervention, such as two or moreparameters, such as three or more parameters, such as four or moreparameters and including five or more parameters. In some embodiments,methods may include adjusting the hydrostatic pressure, the sheath fluidpressure, drop charge, deflection voltage, charge correction value, dropdelay, drop drive frequency, drop amplitude charge phase and anycombinations thereof.

In practicing methods according to certain embodiments, one or moreimages of a flow cytometer flow stream are captured in a detectionfield. As discussed above, the detection fields may vary depending onthe properties of the flow stream being interrogated. In embodiments,methods may include capturing in an image a detection field that spans0.001 mm or more of the flow stream, such as 0.005 mm or more, such as0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, suchas 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 5mm or more and including 10 mm or more of the flow stream. The detectionfield interrogated may vary. In some embodiments, the detection fieldincludes the flow cell nozzle orifice. In other embodiments, thedetection field includes the location of the flow stream where the dropscontaining the particles of interest are charged (i.e., the “break-off”point where the continuous flow stream begins to form discretedroplets). In yet other embodiments, the detection field includes theregion where charged particles are deflected by deflector plates duringcell sorting.

In capturing one or more images of the flow stream, a detection field isilluminated with a light source. In some embodiments, the flow stream isilluminated with a broadband light source or with a narrow band of light(as described above). Suitable broadband light source protocol mayinclude, but are not limited to a halogen lamp, deuterium arc lamp,xenon arc lamp, stabilized fiber-coupled broadband light source, abroadband LED with continuous spectrum, superluminescent emitting diode,semiconductor light emitting diode, wide spectrum LED white lightsource, an multi-LED integrated white light source, among otherbroadband light sources or any combination thereof. Suitable narrow bandlight sources, include but are not limited to a narrow wavelength LED,laser diode or a broadband light source coupled to one or more opticalbandpass filters, diffraction gratings, monochromators or anycombination thereof.

In certain embodiments, the light source is a stroboscopic light sourcewhere the flow stream is illuminated with periodic flashes of light. Forexample, the frequency of light strobe may be 0.01 kHz or greater, suchas 0.05 kHz or greater, such as 0.1 kHz or greater, such as 0.5 kHz orgreater, such as 1 kHz or greater, such as 2.5 kHz or greater, such as 5kHz or greater, such as 10 kHz or greater, such as 25 kHz or greater,such as 50 kHz or greater and including 100 kHz or greater. In someinstances, the strobe frequency is synchronized with droplet drivefrequency. In other instances, the strobe frequency is synchronized withimage capture.

Capturing one or more images of the flow stream may include illuminatingthe flow stream with a combination of light sources, such as with two ormore light sources, such as three or more light sources, such as four ormore light sources and including five or more light sources. Where morethan one light source is employed, the flow stream may be illuminatedwith the light sources simultaneously or sequentially, or a combinationthereof. For example, where images of the flow stream are captured byilluminating with two light sources, the subject methods may includesimultaneously illuminating the flow stream with both light sources. Inother embodiments, capturing images of the flow stream may includesequentially illuminating with two light sources. Where two lightsources are illuminated sequentially, the time each light sourceilluminates the flow stream may independently be 0.001 seconds or more,such as 0.01 seconds or more, such as 0.1 seconds or more, such as 1second or more, such as 5 seconds or more, such as 10 seconds or more,such as 30 seconds or more and including 60 seconds or more. Inembodiments where images of the flow stream are captured by sequentiallyilluminating with two or more light sources, the duration the flowstream is illuminated by each light source may be the same or different.

Images of the flow stream may be captured continuously or in discreteintervals. In some instances, methods include capturing imagescontinuously. In other instances, methods include capturing images indiscrete intervals, such as capturing an image of the flow stream every0.001 millsecond, every 0.01 millsecond, every 0.1 millsecond, every 1millsecond, every 10 millseconds, every 100 millseconds and includingevery 1000 millseconds, or some other interval.

One or more images may be captured in each detection field, such as 2 ormore images of the flow stream in each detection field, such as 3 ormore images, such as 4 or more images, such as 5 or more images, such as10 or more images, such as 15 or more images and including 25 or moreimages. Where more than one image is captured in each detection field,the plurality of images may be automatically stitched together by aprocessor having digital image processing algorithm.

Images of the flow stream in each detection field may be captured at anysuitable distance from the flow stream so long as a usable image of theflow stream is captured. For example, images in each detection field maycaptured at 0.01 mm or more from the flow stream, such as 0.05 mm ormore, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm ormore, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm ormore, such as 15 mm or more, such as 25 mm or more and including 50 mmor more from the flow cytometer flow stream. Images of the flow streamin each detection field may also be captured at any angle from the flowstream. For example, images in each detection field may captured at anangle with respect to the axis of the flow stream which ranges from 10°to 90°, such as from 15° to 85°, such as from 20° to 80°, such as from25° to 75° and including from 30° to 60°. In certain embodiments, imagesin each detection field may captured at a 90° angle with respect to theaxis of the flow stream.

In some embodiments, capturing images of the flow stream include movingone or more imaging sensors alongside the path of the flow stream. Forinstance, the imaging sensor may be moved upstream or downstreamalongside the flow stream capturing images in a plurality of detectionfields. For example, methods may include capturing images of the flowstream in two or more different detection fields, such as 3 or moredetection fields, such as 4 or more detection fields and including 5 ormore detections fields. The imaging sensor may be moved continuously orin discrete intervals. In some embodiments, the imaging sensor is movedcontinuously. In other embodiments, the imaging sensor may be movedalong the flow stream path in discrete intervals, such as for example in1 mm or greater increments, such as 2 mm or greater increments andincluding 5 mm or greater increments.

As summarized above, methods include generating a data signalcorresponding to one or more properties of the flow stream from thecaptured images. In detection fields where the flow stream iscontinuous, methods may include generating a data signal correspondingto the spatial position of the flow stream, the dimensions of the flowstream such as flow stream width, as well as flow rate and flowturbulence based on the captured images. In detection fields where theflow stream is composed of discrete droplets, methods may includegenerating a data signal corresponding to the spatial position of theflow stream, drop size including drop diameter and volume, drop drivefrequency, drop amplitude as well as the uniformity of drop size andfrequency. In certain embodiments, methods include generating a datasignal corresponding to the ratio of the size of the flow stream ascompared to the expected size of the flow stream based on empiricalcharacteristics of the flow cytometer and user inputted data. In otherembodiments, methods include assessing the captured images to determinewhether a flow stream is present or absent in a particular detectionfield. In yet other embodiments, methods include assessing the capturedimages of the flow stream to determine the flow cell nozzle orificesize.

In some embodiments, methods include capturing one or more images of aflow stream of the flow cytometer in a detection field, determining thespatial position of the flow stream in the detection field based on thecaptured images and generating a data signal corresponding to thespatial position of the flow stream. For instance, methods may includecapturing images of the flow stream in a detection field and mapping thespatial position of the flow stream in an X-Y plane. In some instances,the position of the flow stream in the X-Y plane is compared to thevertical axis of the flow cell nozzle to determine position of the flowstream with respect to the vertical axis formed by the flow cell nozzle.Where the spatial position of the flow stream is determined in more thanone detection field, the spatial position of the flow stream may bemapped in an X-Y plane in each detection field and compared to fine tunethe precise spatial position of the flow stream in the X-Y plane. Basedon the determined spatial position of the flow stream in the detectionfield, methods may include generating a data signal corresponding to thespatial position of the flow stream.

In embodiments according to the subject methods, one or more parametersof the flow cytometer are adjusted in response to the data signalcorresponding to the spatial position of the flow stream. In someinstances, the data signal is used to adjust the position of a supportstage having containers for collecting particles, such as during cellsorting. In certain embodiments, methods include generating a datasignal corresponding to the spatial position of the flow stream andautomatically adjusting the position of a support stage so that thecollection containers on the support stage are aligned with thetrajectory of the flow stream. For example, methods may include mappingthe position of the flow stream in each detection field in an X-Y plane,mapping the position of the container in the X-Y plane and matching theposition of the container in the X-Y plane with the position of the flowstream in the X-Y plane to align the collection container with the flowstream. In some instances, methods include mapping the position of theflow stream in two detection fields. In these instances, the spatialposition of the flow stream is mapped in the first detection field in anX-Y plane and the spatial position of the flow stream is mapped in thesecond detection field in the X-Y plane. Based on the mapped positionsof the flow stream in the first and second detection field, a datasignal is generated corresponding to the spatial position of the flowstream in the flow cytometer.

In these embodiments, a data signal is generated corresponding to thespatial position of the flow stream and automatically adjusting theposition of a support stage in an X-Y plane so as to optimize collectionof the flow stream. For example, optimizing collection of the particlesmay include reducing the number of particles not collected by thecontainers on the support stage due to misalignment of the flow streamwith the collection containers, such as by 5% or more as comparedcollecting the flow stream in a container on a support stage notadjusted in response to the data signal, such as by 10% or more, such as15% or more, such as 20% or more, such as 25% or more, such as 35% ormore, such as 50% or more, such as 75% or more, such as 90% or more,such as 95% or more and including by 99% or more.

As described above, support stages may be positioned anywhere along theflow stream as desired when collecting particles from the flow stream.In some instances, particles are collected into containers on a supportstage positioned downstream from deflector plates where the flow streamdroplets have been separated based on charge (e.g., positive, negativeand neutral). In these instances, methods include capturing images of aflow stream in a detection field downstream from the deflector platesand generating a data signal corresponding to the spatial positions ofthe flow streams of the positive, negative and neutral particles. Basedon the determined spatial positions of the flow streams from thecaptured images, the position of a support stage having amulti-compartment container (or three separate containers) may beautomatically adjusted to optimize collection of each flow stream. Forexample, methods may include adjusting the position of the support stagesuch that collection of the flow streams is improved by 5% or more ascompared collecting the flow streams on a support stage not adjusted inresponse to the data signal, such as by 10% or more, such as 15% ormore, such as 20% or more, such as 25% or more, such as 35% or more,such as 50% or more, such as 75% or more, such as 90% or more, such as95% or more and including by 99% or more.

Methods according to certain embodiments are outlined in the combinationof steps shown in FIG. 2. The steps of this invention may occur in anyorder or in any combination. For example in FIG. 2, an experimentalset-up may include the installation of a nozzle appropriate for thesorting task desired. The flow stream may be initiated and an imagecollected of the flow stream by camera 1. The nozzle opening may bedetermined from the image of the flow stream and any number ofparameters may be automatically determined and set based on this value.The laser may be automatically and roughly aligned in accordance withthe signal from the first camera. As the flow stream flows past camera2, a second image may be captured. The two images from camera 1 andcamera 2 may provide for precise localization of the flow stream in anXY plane. A laser may be automatically and finely positioned based onthis information and a collection vessel may be automatically positionedbased on this information. In some embodiments the fine alignment of thelaser may facilitate the alignment of the collection vessel.

As summarized above, methods according to embodiments of the presentdisclosure include adjusting one or more parameters of the flowcytometer in response to data signals derived from captured images inone or more detection fields of a flow cytometer flow stream. In certainembodiments, methods include adjusting sheath fluid pressure, dropletcharging voltage, deflection plate voltage, charge correction value,drop delay, drop drive frequency, drop amplitude and charge phase or acombination thereof.

In some embodiments, methods including adjusting the sheath fluidpressure in response to a data signal corresponding to one or moreproperties of the flow stream determined based on the captured images.In some instances, the sheath fluid pressure may be increased such as by0.001 psi or more, such as 0.005 psi or more, such as by 0.01 psi ormore, such as by 0.05 psi or more, such as by 0.1 psi or more, such as0.5 psi or more, such as by 1 psi or more, such as by 5 psi or more,such as by 10 psi or more, such as by 25 psi or more, such as by 50 psior more, such as by 75 psi or more and including increasing thehydrostatic pressure by 100 psi or more. In other instances, the sheathfluid pressure is reduced, such as by 0.001 psi or more, such as 0.005psi or more, such as by 0.01 psi or more, such as by 0.05 psi or more,such as by 0.1 psi or more, such as 0.5 psi or more, such as by 1 psi ormore, such as by 5 psi or more, such as by 10 psi or more, such as by 25psi or more, such as by 50 psi or more, such as by 75 psi or more andincluding reducing the hydrostatic pressure by 100 psi or more.

In yet other embodiments, methods include adjusting the drop chargingvoltage in response to a data signal corresponding to one or moreproperties of the flow stream determined based on the captured images.In some instances, the drop charging voltage is increased, such as by0.01 V or more, such as 0.05 V or more, such as 0.1 V or more, such asby 0.5V or more, such as by 1V or more, such as by 5V or more, such asby 10V or more, such as by 15V or more, such as by 25V or more, such asby 50V or more and including increasing the drop charging voltage by 75Vor more. In other instances, the drop charging voltage is reduced, suchas by 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more,such as by 0.5V or more, such as by 1V or more, such as by 5V or more,such as by 10V or more, such as by 15V or more, such as by 25V or more,such as by 50V or more and including reducing the drop charging voltageby 75V or more.

In yet other embodiments, methods include adjusting the deflection platevoltage in response to a data signal corresponding to one or moreproperties of the flow stream determined based on the captured images.In some instances, the deflection plate voltage is increased, such as by5V or more, such as by 10V or more, such as by 50V or more, such as by100V or more, such as by 250V or more, such as by 500V or more, such asby 1000V or more and including increasing the deflection plate voltageby 2000V or more. In other instances, the drop charging voltage isreduced, such as by 5V or more, such as by 10V or more, such as by 50Vor more, such as by 100V or more, such as by 250V or more, such as by500V or more, such as by 1000V or more and including reducing thedeflection plate voltage by 2000V or more.

In still other embodiments, methods include adjusting the drop drivefrequency in response to a data signal corresponding to one or moreproperties of the flow stream determined based on the captured images.In some instances, the drop drive frequency is increased, such as by0.01 Hz or more, such as by 0.05 Hz or more, such as by 0.1 Hz or more,such as by 0.25 Hz or more, such as by 0.5 Hz or more, such as by 1 Hzor more, such as by 2.5 Hz or more, such as by 5 Hz or more, such as by10 Hz or more and including by 25 Hz or more. In other instances, thedrop frequency is reduced, such as by 0.01 Hz or more, such as by 0.05Hz or more, such as by 0.1 Hz or more, such as by 0.25 Hz or more, suchas by 0.5 Hz or more, such as by 1 Hz or more, such as by 2.5 Hz ormore, such as by 5 Hz or more, such as by 10 Hz or more and including by25 Hz or more.

In still other embodiments, methods include adjusting the drop delay inresponse to a data signal corresponding to one or more properties of theflow stream determined based on the captured images. In some instances,the drop delay is increased, such as by 0.01 microseconds or more, suchas by 0.05 microseconds or more, such as by 0.1 microseconds or more,such as by 0.3 microseconds or more, such as by 0.5 microseconds ormore, such as by 1 microseconds or more, such as by 2.5 microseconds ormore, such as by 5 microseconds or more, such as by 7.5 microseconds ormore and including increasing the drop delay by 10 microseconds or more.In other instances, the drop frequency is reduced, such as by 0.01microseconds or more, such as by 0.05 microseconds or more, such as by0.1 microseconds or more, such as by 0.3 microseconds or more, such asby 0.5 microseconds or more, such as by 1 microseconds or more, such asby 2.5 microseconds or more, such as by 5 microseconds or more, such asby 7.5 microseconds or more and including reducing the drop delay by 10microseconds or more.

In still other embodiments, methods include adjusting the drop amplitudein response to a data signal corresponding to one or more properties ofthe flow stream determined based on the captured images. In someinstances, the drop amplitude is increased, such as by 0.01 volts ormore, such as by 0.025 volts or more, such as by 0.05 volts or more,such as by 0.1 volts or more, such as by 0.25 volts or more, such as by0.5 volts or more and including increasing the drop amplitude by 1 voltor more. In other instances, the drop amplitude is reduced, such as by0.01 volts or more, such as by 0.025 volts or more, such as by 0.05volts or more, such as by 0.075 volts or more, such as by 0.1 volts ormore, such as by 0.25 volts or more and including reducing the dropamplitude by 1 volt or more.

In some embodiments, methods include capturing one or more images of aflow stream of the flow cytometer in a detection field, characterizingthe physical dimensions of the flow stream in the detection field basedon the captured images and generating a data signal corresponding to thephysical dimensions of the flow stream. In detection fields where theflow stream is a continuous stream, methods may include taking thecaptured images and generating a data signal corresponding to the widthof the flow stream. In detection fields where the flow stream iscomposed of discrete droplets, methods may include taking the capturedimages and generating data signals corresponding to droplet size, suchas droplet diameter.

In certain instances, methods may include determining a flow cell nozzleorifice diameter based on the captured images. In these instances,methods may include capturing images of the flow stream at the orificeof the flow cell nozzle and generating a data signal corresponding tothe physical dimensions of the flow stream. Based on the data signalcorresponding to the physical dimensions of the flow stream, the flowcell nozzle opening diameter is determined. In certain instances,methods include determining the flow cell nozzle opening diameter usingthe width of the flow stream. In other instances, methods includedetermining the flow cell nozzle opening diameter using the dropletdiameter. In these embodiments, methods may further include automatingadjustments to one or more parameters of the flow cytometer based thedetermined flow cell nozzle opening diameter. For example, methods mayinclude automatically adjusting the sheath fluid pressure, drop drivefrequency, drop charge, deflection voltage, charge correction value,drop delay, drop frequency, drop amplitude charge phase and or acombination thereof, as discussed above.

In certain embodiments, methods may include automating adjustments tothe drop drive frequency in response to the determined flow cell nozzleorifice diameter. For example, the drop drive frequency may be increasedby 0.01 Hz or more, such as by 0.05 Hz or more, such as by 0.1 Hz ormore, such as by 0.25 Hz or more, such as by 0.5 Hz or more, such as by1 Hz or more, such as by 2.5 Hz or more, such as by 5 Hz or more, suchas by 10 Hz or more and including by 25 Hz or more. In other instances,methods include reducing the drop drive frequency in response to thedetermined flow cell nozzle orifice diameter, such as by 0.01 Hz ormore, such as by 0.05 Hz or more, such as by 0.1 Hz or more, such as by0.25 Hz or more, such as by 0.5 Hz or more, such as by 1 Hz or more,such as by 2.5 Hz or more, such as by 5 Hz or more, such as by 10 Hz ormore and including by 25 Hz or more.

In other embodiments, methods may include automating adjustments to thesheath fluid pressure in response to the determined flow cell nozzleorifice diameter. For example, the sheath fluid pressure may beincreased by 0.001 psi or more, such as 0.005 psi or more, such as by0.01 psi or more, such as by 0.05 psi or more, such as by 0.1 psi ormore, such as 0.5 psi or more, such as by 1 psi or more, such as by 5psi or more, such as by 10 psi or more, such as by 25 psi or more, suchas by 50 psi or more, such as by 75 psi or more and including increasingthe sheath fluid pressure by 100 psi or more. In other instances,methods include reducing the sheath fluid pressure in response to thedetermined flow cell nozzle orifice diameter, such as by 0.001 psi ormore, such as 0.005 psi or more, such as by 0.01 psi or more, such as by0.05 psi or more, such as by 0.1 psi or more, such as 0.5 psi or more,such as by 1 psi or more, such as by 5 psi or more, such as by 10 psi ormore, such as by 25 psi or more, such as by 50 psi or more, such as by75 psi or more and including reducing the sheath fluid pressure by 100psi or more.

In some embodiments, methods may include comparing the physicaldimensions of the flow stream determined from the captured images withdimensions expected based on empirical characteristics of the flowcytometer (such as flow cell nozzle orifice size and sheath fluidpressure) as well as inputted parameters by the user. In theseinstances, methods include generating a data signal corresponding to theratio of the physical dimensions of the flow stream determined from thecaptured images as compared to the expected flow stream dimensions basedon the empirical characteristics of the flow cytometer and user inputtedparameters. For example, methods may include generating a data signalwhich indicates that the flow stream as determined from the capturedimages is 99% or less of the expected size of the flow stream, such as95% or less, such as 90% or less, such as 85% or less, such as 80% orless, such as 75% or less, such as 50% or less, such as 25% or less andincluding 10% or less of the expected size of the flow stream. In otherembodiments, methods may include generating a data signal whichindicates that the flow stream as determined from the captured images isgreater than the size expected, such as being 105% or greater of thesize of the flow stream, such a 110% or greater, such as 125% or greaterand including 150% or greater. In these embodiments, methods may furtherinclude automating adjustments to one or more parameters of the flowcytometer based on the generated data signal. For example, methods mayinclude automatically adjusting the sheath fluid pump rate, the sheathfluid pressure or drop drive frequency.

Methods of interest may also include capturing images in a detectionfield the break-off point of the flow stream, generating a data signalcorresponding to drop volume of droplets downstream from the break-offpoint and adjusting one or more properties of the flow cytometer inresponse to the determined drop volume. In some embodiments, methodsinclude automatically adjusting the drop drive frequency of the flowstream in response to the determined drop volume. For example, methodsmay include reducing the drop drive frequency, such as by 5% or more,such as by 10% or more, such as by 15% or more, such as by 25% or more,such as by 50% or more, such as by 75% or more, such as by 90% or more,such as by 95% or more and including by 99% or more. In other instances,methods may include increasing the drop drive frequency in response tothe determined drop volume, such as by 5% or more, such as by 10% ormore, such as by 15% or more, such as by 25% or more, such as by 50% ormore, such as by 75% or more, such as by 90% or more, such as by 95% ormore and including by 99% or more.

In other embodiments, methods include automatically regulating samplecollection volume during cell sorting based on the determined dropvolume. For example, the volume desired for each collected sample may beinput into a processor and based on the data signal corresponding to thedrop volume, the flow cytometer may be automated to stop collection ofthe sample after a predetermined amount of time, such as by removing thecollection container or by ceasing the flow stream by the flowcytometer.

In yet other embodiments, methods may include capturing images of a flowstream in a detection field, determining the presence or absence of aflow stream in the detection field and adjusting one or more parametersof the flow cytometer in response to the determined presence or absenceof the flow stream. As discussed above, the subject methods fordetermining whether a flow stream is present or not present in capturedimages of the flow cell nozzle orifice may be used to determine whetherthe flow cell has a clogged nozzle. In these embodiments, capturedimages by the imaging sensors are assessed and if a flow stream isdetected in the images, a data signal is generated indicating thepresence of a flow stream. On the other hand, if after assessing thecaptured images, it is determined that the flow stream is absent in thecaptured images, a data signal is generated indicating the absence of aflow stream.

Where no flow stream is present in the captured images, in certainembodiments, methods may include automatically alerting a user that theabsence of flow stream is a result of flow cytometer malfunction, suchas a clogged nozzle. In these embodiments, data signal corresponding tothe absence of a flow stream is correlated with input from the user asto whether a flow stream should be expected. In some embodiments, wherea user has configured the system to have a “closed loop” configuration,no flow stream is expected. In these embodiments, the flow cytometerdoes not alert the user of a malfunction (e.g., clogged nozzle) since aflow stream is not expected.

FIG. 3 depicts a flow chart illustrating methods for adjusting one ormore parameters of a flow cytometer according to certain embodiments ofthe present disclosure. As summarized above, methods include capturingone or more images in a detection field of a flow cytometer flow stream.The images may, in certain instances, be captured in two or moredetection fields, such as 3 or more and including 4 or more detectionfields. In some embodiments, methods include determining whether a flowstream is present or absent. Where a flow stream is determined to beabsent and a flow stream is expected (such as during normal usage), analert may be conveyed to the user of a possible instrument malfunction(e.g., clogged nozzle). In other embodiments, methods includedetermining the spatial position of the flow stream or determining thephysical dimensions of the flow stream. In certain instances, methodsinclude initially determining that a flow stream is present in the oneor more captured images, followed by determining the spatial position ofthe flow stream. In other instances, methods include initiallydetermining that a flow stream is present in the one or more capturedimages, followed by determining the physical dimensions of the flowstream. In some embodiments, methods including determining a physicalproperty of the flow cytometer based on the physical dimensions of theflow stream from the captured images. For example, the flow cell nozzleorifice may be determined based on the physical dimensions of the flowstream from the captured images.

Methods also include automatically adjusting one or more parameters ofthe flow cytometer in response to data signals derived from capturedimages, such as adjusting sheath fluid pressure, droplet chargingvoltage, deflection plate voltage, charge correction value, drop delay,drop drive frequency, drop amplitude and charge phase or a combinationthereof. In certain embodiments, the one or more parameters includesadjusting the position of one or more support stages, for example, asupport stage having a container for collecting flow stream particlesduring cell sorting.

As discussed above, the subject methods may be fully automated, suchthat adjustments are made in response to data signals corresponding toone or more parameters of the flow stream with little, if any, humanintervention or manual input by the user.

Computer-Controlled Systems

Aspects of the present disclosure further include computer controlledsystems for practicing the subject methods, where the systems furtherinclude one or more computers for complete automation or partialautomation of a system for practicing methods described herein. In someembodiments, systems include a computer having a computer readablestorage medium with a computer program stored thereon, where thecomputer program when loaded on the computer includes instructions forcapturing one or more images of a flow stream of the flow cytometer in adetection field; algorithm for determining the spatial position of theflow stream in the detection field; algorithm for generating a datasignal corresponding to the spatial position of the flow stream; andinstructions for adjusting one or more parameters of the flow cytometerin response to the data signal. In certain instances, systems include acomputer having a computer readable storage medium with a computerprogram stored thereon, where the computer program when loaded on thecomputer includes instructions for capturing one or more images of aflow stream of the flow cytometer in a detection field; algorithm fordetermining the physical dimensions of the flow stream in the detectionfield; algorithm for generating a data signal corresponding to thephysical dimensions of the flow stream; and instructions for adjustingone or more parameters of the flow cytometer in response to the datasignal.

In embodiments, the system includes an input module, a processing moduleand an output module. Processing modules of interest may include one ormore processors that are configured and automated to adjust one or moreparameters of a flow cytometer as described above. For exampleprocessing modules may include two or more processors that areconfigured and automated to adjust one or more parameters of a flowcytometer as described above, such as three or more processors, such asfour or more processors and including five or more processors.

In some embodiments, the subject systems may include an input modulesuch that parameters or information about the fluidic sample, sheathfluid pressure, hydrostatic pressure, flow stream charge, deflectionvoltage, charge correction value, drop delay, drop drive frequency, dropamplitude and charge phase, flow cell nozzle orifice, position ofsupport stages, imaging sensors, light sources, optical adjustmentprotocols, amplifiers as well as properties, resolution and sensitivityof imaging sensors may be input before practicing the subject methods.

As described above, each processor includes memory having a plurality ofinstructions for performing the steps of the subject methods, such ascapturing one or more images of a flow stream of the flow cytometer in adetection field; determining one or more properties of the flow streamin the detection field; generating a data signal corresponding to theone or more properties of the flow stream; and adjusting one or moreparameters of the flow cytometer in response to the data signal. Afterthe processor has performed one or more of the steps of the subjectmethods, the processor may be automated to make adjustments toparameters of the flow cytometer, such as adjustments as describedabove.

The subject systems may include both hardware and software components,where the hardware components may take the form of one or moreplatforms, e.g., in the form of servers, such that the functionalelements, i.e., those elements of the system that carry out specifictasks (such as managing input and output of information, processinginformation, etc.) of the system may be carried out by the execution ofsoftware applications on and across the one or more computer platformsrepresented of the system.

Systems may include a display and operator input device. Operator inputdevices may, for example, be a keyboard, mouse, or the like. Theprocessing module includes a processor which has access to a memoryhaving instructions stored thereon for performing the steps of thesubject methods. The processing module may include an operating system,a graphical user interface (GUI) controller, a system memory, memorystorage devices, and input-output controllers, cache memory, a databackup unit, and many other devices. The processor may be a commerciallyavailable processor or it may be one of other processors that are orwill become available. The processor executes the operating system andthe operating system interfaces with firmware and hardware in awell-known manner, and facilitates the processor in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages, such as Java, Perl, C++, otherhigh level or low level languages, as well as combinations thereof, asis known in the art. The operating system, typically in cooperation withthe processor, coordinates and executes functions of the othercomponents of the computer. The operating system also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, flash memorydevices, or other memory storage device. The memory storage device maybe any of a variety of known or future devices, including a compact diskdrive, a tape drive, a removable hard disk drive, or a diskette drive.Such types of memory storage devices typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, or floppy diskette.Any of these program storage media, or others now in use or that maylater be developed, may be considered a computer program product. Aswill be appreciated, these program storage media typically store acomputer software program and/or data. Computer software programs, alsocalled computer control logic, typically are stored in system memoryand/or the program storage device used in conjunction with the memorystorage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor the computer, causes the processor to performfunctions described herein. In other embodiments, some functions areimplemented primarily in hardware using, for example, a hardware statemachine. Implementation of the hardware state machine so as to performthe functions described herein will be apparent to those skilled in therelevant arts.

Memory may be any suitable device in which the processor can store andretrieve data, such as magnetic, optical, or solid state storage devices(including magnetic or optical disks or tape or RAM, or any othersuitable device, either fixed or portable). The processor may include ageneral purpose digital microprocessor suitably programmed from acomputer readable medium carrying necessary program code. Programmingcan be provided remotely to processor through a communication channel,or previously saved in a computer program product such as memory or someother portable or fixed computer readable storage medium using any ofthose devices in connection with memory. For example, a magnetic oroptical disk may carry the programming, and can be read by a diskwriter/reader. Systems of the invention also include programming, e.g.,in the form of computer program products, algorithms for use inpracticing the methods as described above. Programming according to thepresent invention can be recorded on computer readable media, e.g., anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM;portable flash drive; and hybrids of these categories such asmagnetic/optical storage media.

The processor may also have access to a communication channel tocommunicate with a user at a remote location. By remote location ismeant the user is not directly in contact with the system and relaysinput information to an input manager from an external device, such as aa computer connected to a Wide Area Network (“WAN”), telephone network,satellite network, or any other suitable communication channel,including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may beconfigured to include a communication interface. In some embodiments,the communication interface includes a receiver and/or transmitter forcommunicating with a network and/or another device. The communicationinterface can be configured for wired or wireless communication,including, but not limited to, radio frequency (RF) communication (e.g.,Radio-Frequency Identification (RFID), Zigbee communication protocols,WiFi, infrared, wireless Universal Serial Bus (USB), Ultra Wide Band(UWB), Bluetooth® communication protocols, and cellular communication,such as code division multiple access (CDMA) or Global System for Mobilecommunications (GSM).

In one embodiment, the communication interface is configured to includeone or more communication ports, e.g., physical ports or interfaces suchas a USB port, an RS-232 port, or any other suitable electricalconnection port to allow data communication between the subject systemsand other external devices such as a computer terminal (for example, ata physician's office or in hospital environment) that is configured forsimilar complementary data communication.

In one embodiment, the communication interface is configured forinfrared communication, Bluetooth® communication, or any other suitablewireless communication protocol to enable the subject systems tocommunicate with other devices such as computer terminals and/ornetworks, communication enabled mobile telephones, personal digitalassistants, or any other communication devices which the user may use inconjunction.

In one embodiment, the communication interface is configured to providea connection for data transfer utilizing Internet Protocol (IP) througha cell phone network, Short Message Service (SMS), wireless connectionto a personal computer (PC) on a Local Area Network (LAN) which isconnected to the internet, or WiFi connection to the internet at a WiFihotspot.

In one embodiment, the subject systems are configured to wirelesslycommunicate with a server device via the communication interface, e.g.,using a common standard such as 802.11 or Bluetooth® RF protocol, or anIrDA infrared protocol. The server device may be another portabledevice, such as a smart phone, Personal Digital Assistant (PDA) ornotebook computer; or a larger device such as a desktop computer,appliance, etc. In some embodiments, the server device has a display,such as a liquid crystal display (LCD), as well as an input device, suchas buttons, a keyboard, mouse or touch-screen.

In some embodiments, the communication interface is configured toautomatically or semi-automatically communicate data stored in thesubject systems, e.g., in an optional data storage unit, with a networkor server device using one or more of the communication protocols and/ormechanisms described above.

Output controllers may include controllers for any of a variety of knowndisplay devices for presenting information to a user, whether a human ora machine, whether local or remote. If one of the display devicesprovides visual information, this information typically may be logicallyand/or physically organized as an array of picture elements. A graphicaluser interface (GUI) controller may include any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between the system and a user, and for processing userinputs. The functional elements of the computer may communicate witheach other via system bus. Some of these communications may beaccomplished in alternative embodiments using network or other types ofremote communications. The output manager may also provide informationgenerated by the processing module to a user at a remote location, e.g.,over the Internet, phone or satellite network, in accordance with knowntechniques. The presentation of data by the output manager may beimplemented in accordance with a variety of known techniques. As someexamples, data may include SQL, HTML or XML documents, email or otherfiles, or data in other forms. The data may include Internet URLaddresses so that a user may retrieve additional SQL, HTML, XML, orother documents or data from remote sources. The one or more platformspresent in the subject systems may be any type of known computerplatform or a type to be developed in the future, although theytypically will be of a class of computer commonly referred to asservers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known orfuture type of cabling or other communication system including wirelesssystems, either networked or otherwise. They may be co-located or theymay be physically separated. Various operating systems may be employedon any of the computer platforms, possibly depending on the type and/ormake of computer platform chosen. Appropriate operating systems includeWindows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux,OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others.

Utility

The subject systems, methods, and computer systems find use in a varietyof different applications where it is desirable to automate adjustmentsto one or more parameters of a flow cytometer to provide for fast,reliable systems for characterizing and sorting cells from a biologicalsample. Embodiments of the present disclosure find use where minimizingthe amount of reliance on human input and adjustments to the system aredesired, such as in research and high throughput laboratory testing. Thepresent disclosure also finds use where it is desirable to provide aflow cytometer with improved cell sorting accuracy, enhanced particlecollection, systems which provide alerts regarding component malfunction(e.g., clogged flow cell nozzle), reduced energy consumption, particlecharging efficiency, more accurate particle charging and enhancedparticle deflection during cell sorting. In embodiments, the presentdisclosure reduces the need for user input or manual adjustment duringsample analysis with a flow cytometer. In certain embodiments, thesubject systems provide fully automated protocols so that adjustments toa flow cytometer during use require little, if any human input.

The present disclosure also finds use in applications where cellsprepared from a biological sample may be desired for research,laboratory testing or for use in therapy. In some embodiments, thesubject methods and devices may facilitate the obtaining individualcells prepared from a target fluidic or tissue biological sample. Forexample, the subject methods and systems facilitate obtaining cells fromfluidic or tissue samples to be used as a research or diagnosticspecimen for diseases such as cancer. Likewise, the subject methods andsystems facilitate obtaining cells from fluidic or tissue samples to beused in therapy. Methods and devices of the present disclosure allow forseparating and collecting cells from a biological sample (e.g., organ,tissue, tissue fragment, fluid) with enhanced efficiency and low cost ascompared to traditional flow cytometry systems.

Notwithstanding the appended clauses, the disclosure set forth herein isalso defined by the following clauses:1. A system comprising:

an imaging sensor configured to capture one or more images of a flowstream in a detection field of a flow cytometer; and

a processor comprising memory operably coupled to the processor, whereinthe memory includes instructions stored thereon to determine one or moreproperties of the flow stream and generate a data signal correspondingto the one or more properties of the flow stream,

wherein the processor is configured to automatically adjust one or moreparameters of the flow cytometer in response to data signal.

2. The system according to clause 1, wherein the processor is configuredto determine the spatial position of the flow stream and generate a datasignal corresponding to the spatial position of the flow stream from theone or more images.3. The system according to any of clauses 1-2, wherein the systemfurther comprises a support stage positioned downstream from thedetection field.4. The system according to clause 3, wherein the system is configured toautomatically adjust the position of the support stage in response tothe data signal corresponding to the spatial position of the flow streamin the detection field.5. The system according to clause 4, wherein the system is configured toadjust the position of the support stage in two dimensions.6. The system according to any of clauses 3-5, wherein the support stagecomprises a laser.7. The system according to any of clauses 3-5, wherein the support stagecomprises a container.8. The system according to clause 7, wherein the system is configured toautomatically align the container with the determined spatial positionof the flow stream.9. The system according to clause 8, wherein automatically aligning thecontainer with the flow stream comprises:

mapping the position of the flow stream in the detection field in an X-Yplane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

10. The system according to any of clauses 1-9, wherein the systemfurther comprises:

a second imaging sensor configured to capture one or more images of theflow stream in a second detection field; and

a processor comprising memory operably coupled to the processor, whereinthe memory includes instructions stored thereon to determine one or moreproperties of the flow stream in the second detection field and generatea second data signal corresponding to the one or more properties of theflow stream in the second detection field.

11. The system according to clause 10, wherein the processor isconfigured to determine the spatial position of the flow stream in thesecond detection field and generate a second data signal correspondingto the spatial position of the flow stream in the second detectionfield.12. The system according to clause 11, wherein the system furthercomprises a second support stage positioned downstream from the firstsupport stage.13. The system according to clause 12, wherein the system is configuredto automatically adjust the position of the second support stage inresponse to the first and second data signals.14. The system according to clause 13, wherein the system is configuredto adjust the position of the second support stage in two dimensions.15. The system according to clause 13, wherein the second support stagecomprises a container for collecting the flow stream.16. The system according to clause 15, wherein the system is configuredto automatically align the container with the determined spatialposition of the flow stream in the second detection field.17. The system according to clause 16, wherein automatically aligningthe container comprises:

mapping the position of the flow stream in the second detection field inan X-Y plane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

18. The system according to clause 1, wherein the processor isconfigured to determine the physical dimensions of the flow stream fromthe one or more images and generate a data signal corresponding to thephysical dimensions of the flow stream.19. The system according to clause 18, wherein the processor isconfigured to determine the width of the flow stream from the one ormore images and generate a data signal corresponding to the width of theflow stream.20. The system according to clause 19, wherein the processor isconfigured to determine flow cell nozzle orifice diameter and generate adata signal corresponding to the flow cell nozzle orifice diameter basedon the determined width of the flow stream.21. The system according to clause 20, wherein the processor isconfigured to automatically adjust one or more parameters of the flowcytometer based on the determined flow cell nozzle orifice diameter.22. The system according to clause 21, wherein the parameters of theflow cytometer is selected from the group consisting of hydrostaticpressure, sheath fluid pressure, flow stream charge, deflection voltage,oscillator drive frequency, charge correction value, drop delay, dropfrequency, drop amplitude and charge phase.23. The system according to clause 22, wherein the processor isconfigured to automatically adjust sheath fluid pressure based on thedetermined flow cell nozzle orifice diameter.24. The system according to clause 22, wherein the processor isconfigured to automatically adjust oscillator drive frequency based onthe determined flow cell nozzle orifice diameter.25. The system according to any of clauses 1-24, wherein the imagingsensor is a CCD camera.26. A system for automatically localizing a stream position in a liquidflow from a flow cytometer comprising;

a first camera, adapted to detect a stream position in a first detectionfield and to generate a first signal representative of the streamposition; and

a first stage wherein the first stage is operationally connected to thefirst camera and configured to move in an XY plane in response to thefirst signal.

27. The system of clause 26, further comprising a second camera adaptedto detect a steam position in a second detection field and to generate asecond signal representative of the stream position;

wherein the first and second detection fields of the first and secondcameras are substantially orthogonally oriented in the XY plane; and

wherein the first stage is operationally connected to the second cameraand configured to move the XY plane in response to the second signal inaddition to the first signal.

28. The system according to any of clauses 26-27, wherein a laser ismounted on the first stage.29. The system according to any of clauses 26-27, wherein a collectiondevice is mounted on the first stage.30. The system according to clause 26, further comprising a second stagewherein a collection device is mounted on the second stage and thesecond stage in configured to move in the XY plane in response to thefirst signal.31. The system according to clause 30, further comprising a second stagewherein a collection device is mounted on the second stage the secondstage is configured to move in the XY plane in response to the secondsignal in addition to the first signal.32. The system according to clause 30, further comprising an electricalsystem configured to adjust an electrical charge on the flow stream inresponse to the second signal from the second camera.33. The system according to clause 30, wherein the operationalconnection is mediated by a controller connected to the first camera andthe first and second camera and the first stage and wherein thecontroller is configured to receive the signals from the first andsecond cameras and calculate an optimum position for the first stage.34. The system according to clause 33, wherein the operationalconnection is mediated by a controller connected to the first and secondcamera and the second stage and configured to receive the signals fromthe first and second cameras and calculate an optimum position for thesecond stage.35. The system according to any of clauses 26-34, wherein the stream iscomprised of a series of drops.36. A system for automatically determining a nozzle opening diametercomprising

a first camera, adapted to detect a stream dimension in a firstdetection field and to generate a first signal representative of thestream dimension;

a controller comprising a computer algorithm configured to determine avalue for the nozzle opening diameter from the stream dimension andtransmit the value to a flow cytometer.

37. The system according to clause 36, wherein the stream dimension isthe width of the stream.38. The system according to any of clauses 36-37, wherein the flowcytometer is configured to automatically adjust a series of parametersafter receiving the transmitted value.39. The system according to clause 38, wherein the series of parametersare selected from the group comprising hydrostatic pressure, dropcharge, deflection voltage, charge correction value, drop delay, dropdrive frequency, drop amplitude, and charge phase.40. A method for adjusting one or more parameters of a flow cytometer,the method comprising:

capturing one or more images of a flow cytometer flow stream in adetection field;

determining one or more properties of the flow stream in the detectionfield;

generating a data signal corresponding to the one or more properties ofthe flow stream; and

adjusting one or more parameters of the flow cytometer in response tothe data signal.

41. The method according to clause 40, wherein the method comprisesdetermining the spatial position of the flow stream in the detectionfield and generating a data signal corresponding to the spatial positionof the flow stream.42. The method according to any of clauses 40-41, wherein the flowstream in the detection field is continuous.43. The method according to clause 40, wherein the detection fieldcomprises the flow stream upstream from the flow stream break-off point.44. The method according to clause 43, wherein determining the spatialposition of the flow stream comprises mapping the position of the flowstream in an X-Y plane.45. The method according to clause 44, further comprising adjusting theposition of a support stage in response to the data signal correspondingto the spatial position of the flow stream.46. The method according to clause 45, wherein the support stagecomprises a laser.47. The method according to clause 45, wherein the support stagecomprises a collection container.48. The method according to clause 47, wherein the method comprisesaligning the container with the determined spatial position of the flowstream.47. The method according to clause 48, wherein aligning the containerwith the flow stream comprises:

mapping the position of the flow stream in the detection field in an X-Yplane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

48. The method according any of clauses 40-47, wherein the methodfurther comprises:

capturing one or more images of a flow cytometer flow stream in a seconddetection field;

determining one or more properties of the flow stream in the seconddetection field; and

generating a data signal corresponding to the one or more properties ofthe flow stream in the second detection field.

49. The method according to clause 48, wherein the method comprisesdetermining the spatial position of the flow stream in the seconddetection field and generating a second data signal corresponding to thespatial position of the flow stream in the second detection field.50. The method according to clause 48, wherein the flow stream in thesecond detection stream comprises discrete droplets.51. The method according to clause 48, wherein the second detectionfield comprises the flow stream downstream from the flow streambreak-off point.52. The method according to clause 48, further comprising adjusting theposition of a second support stage in response to the second data signalcorresponding to the spatial position of the flow stream in the seconddetection field.53. The method according to clause 48, wherein the method comprisesadjusting the position of the second support stage in response to thefirst and second data signals.54. The method according to clause 53, wherein the second support stagecomprises a collection container.55. The method according to clause 54, wherein the method comprisesaligning the container with the determined spatial position of the flowstream in the second detection field.56. The method according to clause 55, wherein aligning the containercomprises:

mapping the position of the flow stream in the second detection field inan X-Y plane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

57. The method according to clause 40, wherein the method comprisesdetermining the physical dimensions of the flow stream in the detectionfield and generating a data signal corresponding to the physicaldimensions of the flow stream.58. The method according to clause 57, wherein the method comprisesdetermining the width of the flow stream from the one or more images andgenerating a data signal corresponding to the width of the flow stream.59. The method according to clause 58, further comprising determiningthe flow cell orifice diameter and generating a data signalcorresponding to the flow cell nozzle orifice diameter based on thedetermined width of the flow stream.60. The method according to clause 59, further comprising adjusting oneor more parameters of the flow cytometer based on the determined flowcell nozzle orifice diameter.61. The method according to clause 60, wherein the parameters of theflow cytometer is selected from the group consisting of hydrostaticpressure, sheath fluid pressure, flow stream charge, deflection voltage,oscillator drive frequency, charge correction value, drop delay, dropdrive frequency, drop amplitude and charge phase.62. The method according to clause 61, further comprising adjusting thedrop drive frequency in response to the determined flow cell nozzleorifice diameter.63. The method according to clause 62, further comprising adjusting thesheath fluid pressure in response to the determined flow cell nozzleorifice diameter.64. A method comprising:

capturing one or more images of a flow cytometer flow stream in adetection field;

determining that the flow stream is not present in the captured image;

assessing parameters of the flow cytometer inputted by a user todetermine if the flow stream is expected to be present in the capturedimage; and

generating an alert to the user indicating a flow cytometer malfunction.

65. The method according to clause 64, wherein the malfunction is aclogged nozzle.66. The method according to clause 64, wherein the detection fieldcomprises flow stream upstream from the flow stream break-off point.67. The method according to clause 64, wherein the flow stream in thedetection stream is continuous.68. The method according to clause 64, further comprising inputting thatthe flow cytometer comprises an open flow cell nozzle orifice.69. A method for adjusting one or more parameters of a flow cytometer,the method comprising:

injecting a sample into the sample port of a flow cytometer, wherein theflow cytometer comprises a system comprising a processor with memoryoperably coupled to the processor wherein the system is automated to:

capture one or more images of a flow cytometer flow stream comprisingthe sample in a detection field;

determine one or more properties of the flow stream in the detectionfield;

generate a data signal corresponding to the one or more properties ofthe flow stream; and

adjust one or more parameters of the flow cytometer in response to thedata signal.

70. The method according to clause 69, wherein the method comprisesdetermining the spatial position of the flow stream in the detectionfield and generating a data signal corresponding to the spatial positionof the flow stream.71. The method according to clause 69, wherein the flow stream in thedetection stream is continuous.72. The method according to clause 69, wherein the detection fieldcomprises the flow stream upstream from the flow stream break-off point.73. The method according to clause 72, wherein determining the spatialposition of the flow stream comprises mapping the position of the flowstream in an X-Y plane.74. The method according to clause 69, further comprising adjusting theposition of a support stage in response to the data signal correspondingto the spatial position of the flow stream.75. The method according to clause 74, wherein the support stagecomprises a laser.76. The method according to clause 74, wherein the support stagecomprises a collection container.77. The method according to clause 76, wherein the method comprisesaligning the container with the determined spatial position of the flowstream.78. The method according to clause 77, wherein aligning the containerwith the flow stream comprises:

mapping the position of the flow stream in the detection field in an X-Yplane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

79. The method according clause 69, wherein the method furthercomprises:

capturing one or more images of a flow cytometer flow stream in a seconddetection field;

determining one or more properties of the flow stream in the seconddetection field; and

generating a data signal corresponding to the one or more properties ofthe flow stream in the second detection field.

80. The method according to clause 79, wherein the method comprisesdetermining the spatial position of the flow stream in the seconddetection field and generating a second data signal corresponding to thespatial position of the flow stream in the second detection field.81. The method according to clause 80, wherein the flow stream in thesecond detection stream comprises discrete droplets.82. The method according to clause 80, wherein the second detectionfield comprises the flow stream downstream from the flow streambreak-off point.83. The method according to clause 80, further comprising adjusting theposition of a second support stage in response to the second data signalcorresponding to the spatial position of the flow stream in the seconddetection field.84. The method according to clause 79, wherein the method comprisesadjusting the position of the second support stage in response to thefirst and second data signals.85. The method according to clause 84, wherein the second support stagecomprises a collection container.86. The method according to clause 85, wherein the method comprisesaligning the container with the determined spatial position of the flowstream in the second detection field.87. The method according to clause 86, wherein aligning the containercomprises:

mapping the position of the flow stream in the second detection field inan X-Y plane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

88. The method according to clause 69, wherein the method comprisesdetermining the physical dimensions of the flow stream in the detectionfield and generating a data signal corresponding to the physicaldimensions of the flow stream.89. The method according to clause 88, wherein the method comprisesdetermining the width of the flow stream from the one or more images andgenerating a data signal corresponding to the width of the flow stream.90. The method according to clause 89, further comprising determiningthe flow cell orifice diameter and generating a data signalcorresponding to the flow cell nozzle orifice diameter based on thedetermined width of the flow stream.91. The method according to clause 90, further comprising adjusting oneor more parameters of the flow cytometer based on the determined flowcell nozzle orifice diameter.92. The method according to clause 91, wherein the parameters of theflow cytometer is selected from the group consisting of hydrostaticpressure, sheath fluid pressure, flow stream charge, deflection voltage,oscillator drive frequency, charge correction value, drop delay, dropfrequency, drop amplitude and charge phase.93. The method according to clause 92, further comprising adjusting theoscillator drive frequency in response to the determined flow cellnozzle orifice diameter.94. The method according to clause 92, further comprising adjusting thesheath fluid pressure in response to the determined flow cell nozzleorifice diameter.95. A system for configuring a flow cytometer, the system comprising:

a processor comprising memory operably coupled to the processor, whereinthe memory includes instructions stored thereon, the instructionscomprising:

-   -   instructions for capturing one or more images of a flow        cytometer flow stream in a detection field;    -   algorithm for determining one or more properties of the flow        stream in the detection field;    -   algorithm for generating a data signal corresponding to the one        or more properties of the flow stream; and    -   instructions for adjusting one or more parameters of the flow        cytometer in response to the data signal.        96. The system according to clause 95, wherein the memory        comprises algorithm determining the spatial position of the flow        stream in the detection field and generating a data signal        corresponding to the spatial position of the flow stream.        97. The system according to clause 95, wherein the flow stream        in the detection stream is continuous.        98. The system according to clause 95, wherein the detection        field comprises the flow stream upstream from the flow stream        break-off point.        99. The system according to clause 95, wherein the memory        comprises algorithm for determining the spatial position of the        flow stream comprises mapping the position of the flow stream in        an X-Y plane.        100. The system according to clause 95, wherein the memory        comprises algorithm for adjusting the position of a support        stage in response to the data signal corresponding to the        spatial position of the flow stream.        101. The system according to clause 100, wherein the support        stage comprises a collection container.        102. The system according to clause 101, wherein the memory        comprises algorithm for aligning the container with the        determined spatial position of the flow stream.        103. The system according to clause 102, wherein aligning the        container with the flow stream comprises:

mapping the position of the flow stream in the detection field in an X-Yplane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

104. The system according to clause 95, wherein the memory furthercomprises:

instructions for capturing one or more images of a flow cytometer flowstream in a second detection field;

algorithm for determining one or more properties of the flow stream inthe second detection field; and

algorithm for generating a data signal corresponding to the one or moreproperties of the flow stream in the second detection field.

105. The system according to clause 104, wherein the memory comprisesalgorithm for determining the spatial position of the flow stream in thesecond detection field and generating a second data signal correspondingto the spatial position of the flow stream in the second detectionfield.106. The system according to clause 104, wherein the flow stream in thesecond detection stream comprises discrete droplets.107. The system according to clause 104, wherein the second detectionfield comprises the flow stream downstream from the flow streambreak-off point.108. The system according to clause 104, wherein the memory furthercomprises algorithm for adjusting the position of a second support stagein response to the second data signal corresponding to the spatialposition of the flow stream in the second detection field.109. The system according to clause 104, where the memory comprisesalgorithm for aligning a container on the second support stage with thedetermined spatial position of the flow stream in the second detectionfield.110. The system according to clause 109, wherein aligning the containercomprises:

mapping the position of the flow stream in the second detection field inan X-Y plane;

mapping the position of the container in the X-Y plane; and

matching the position of the container with the position of the flowstream in the X-Y plane.

111. The system according to clause 95, wherein the memory comprisesalgorithm for determining the physical dimensions of the flow stream inthe detection field and generating a data signal corresponding to thephysical dimensions of the flow stream.112. The system according to clause 111, wherein the physical dimensionis the width of the flow stream.113. The system according to clause 111, wherein the memory furthercomprises algorithm for determining the flow cell orifice diameter andgenerating a data signal corresponding to the flow cell nozzle orificediameter based on the determined width of the flow stream.114. The system according to clause 113, wherein the memory furthercomprises algorithm for adjusting one or more parameters of the flowcytometer based on the determined flow cell nozzle orifice diameter.115. The system according to clause 114, wherein the one or moreparameters are selected from the group consisting of hydrostaticpressure, sheath fluid pressure, flow stream charge, deflection voltage,oscillator drive frequency, charge correction value, drop delay, dropfrequency, drop amplitude and charge phase.116. A system for configuring a flow cytometer, the system comprising:

a processor comprising memory operably coupled to the processor, whereinthe memory includes instructions stored thereon, the instructionscomprising:

-   -   instructions for capturing one or more images of a flow        cytometer flow stream in a detection field;    -   algorithm for determining that the flow stream is not present in        the captured image;    -   algorithm for assessing parameters of the flow cytometer        inputted by a user to determine if the flow stream is expected        to be present in the captured image; and    -   instructions for generating an alert to the user indicating a        flow cytometer malfunction.        117. The system according to clause 116, wherein the malfunction        is a clogged nozzle.        118. The system according to clause 116, wherein the detection        field comprises flow stream upstream from the flow stream        break-off point.        119. The system according to clause 116, wherein the flow stream        in the detection stream is continuous.        120. The system according to clause 116, wherein the inputted        parameter comprises indicating that the flow cytometer comprises        an open flow cell nozzle orifice.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

What is claimed is:
 1. A system comprising: an imaging sensor configuredto capture one or more images of a flow stream in a detection field of aflow cytometer; and a processor comprising memory operably coupled tothe processor, wherein the memory includes instructions stored thereonto determine the physical dimensions of the flow stream from the one ormore images and generate a data signal corresponding to the physicaldimensions of the flow stream, wherein the processor is configured toautomatically adjust one or more parameters of the flow cytometer inresponse to data signal.
 2. The system according to claim 1, wherein theprocessor is configured to determine the width of the flow stream fromthe one or more images and generate a data signal corresponding to thewidth of the flow stream.
 3. The system according to claim 2, whereinthe processor is configured to determine flow cell nozzle orificediameter and generate a data signal corresponding to the flow cellnozzle orifice diameter based on the determined width of the flowstream.
 4. The system according to claim 3, wherein the processor isconfigured to automatically adjust one or more parameters of the flowcytometer based on the determined flow cell nozzle orifice diameter. 5.The system according to claim 4, wherein the parameters of the flowcytometer is selected from the group consisting of hydrostatic pressure,sheath fluid pressure, flow stream charge, deflection voltage, dropdrive frequency, charge correction value, drop delay, drop amplitude andcharge phase.
 6. The system according to claim 5, wherein the processoris configured to automatically adjust sheath fluid pressure based on thedetermined flow cell nozzle orifice diameter.
 7. The system according toclaim 5, wherein the processor is configured to automatically adjustdrop drive frequency based on the determined flow cell nozzle orificediameter.
 8. The system according to claim 1, wherein the imaging sensoris a CCD camera.
 9. A system for automatically determining a nozzleopening diameter comprising a first sensor, adapted to detect a streamdimension in a first detection field and to generate a first signalrepresentative of the stream dimension; a controller comprising acomputer algorithm configured to determine a value for the nozzleopening diameter from the stream dimension and transmit the value to aflow cytometer.
 10. The system according to claim 9, wherein the streamdimension is the width of the stream.
 11. The system according to claim9, wherein the flow cytometer is configured to automatically adjust aseries of parameters after receiving the transmitted value.
 12. Thesystem according to claim 9, wherein the series of parameters areselected from the group comprising hydrostatic pressure, drop charge,deflection voltage, charge correction value, drop delay, drop drivefrequency, drop amplitude, and charge phase.
 13. The system according toclaim 12, wherein the parameter is sheath fluid pressure.
 14. The systemaccording to claim 12, wherein the parameter drop drive frequency.
 15. Amethod for adjusting one or more parameters of a flow cytometer, themethod comprising: capturing one or more images of a flow cytometer flowstream in a detection field; determining the physical dimensions of theflow stream in the detection field; generating a data signalcorresponding to the physical dimensions of the flow stream; andadjusting one or more parameters of the flow cytometer in response tothe data signal.
 16. The method according to claim 15, wherein themethod comprises determining the width of the flow stream from the oneor more images and generating a data signal corresponding to the widthof the flow stream.
 17. The method according to claim 16, furthercomprising determining the flow cell orifice diameter and generating adata signal corresponding to the flow cell nozzle orifice diameter basedon the determined width of the flow stream.
 18. The method according toclaim 17, further comprising adjusting one or more parameters of theflow cytometer based on the determined flow cell nozzle orificediameter.
 19. The method according to claim 18, wherein the parametersof the flow cytometer is selected from the group consisting ofhydrostatic pressure, sheath fluid pressure, flow stream charge,deflection voltage, drop drive frequency, charge correction value, dropdelay, drop amplitude and charge phase.
 20. The method according toclaim 19, further comprising adjusting the drop drive frequency inresponse to the determined flow cell nozzle orifice diameter.
 21. Themethod according to claim 19, further comprising adjusting the sheathfluid pressure in response to the determined flow cell nozzle orificediameter.
 22. A method for adjusting one or more parameters of a flowcytometer, the method comprising: injecting a sample into the sampleport of a flow cytometer, wherein the flow cytometer comprises a systemcomprising a processor with memory operably coupled to the processorwherein the system is automated to: capture one or more images of a flowcytometer flow stream comprising the sample in a detection field;determine the physical dimensions of the flow stream in the detectionfield; generate a data signal corresponding to the physical dimensionsof the flow stream; and adjust one or more parameters of the flowcytometer in response to the data signal.
 23. A system for configuring aflow cytometer, the system comprising: a processor comprising memoryoperably coupled to the processor, wherein the memory includesinstructions stored thereon, the instructions comprising: instructionsfor capturing one or more images of a flow cytometer flow stream in adetection field; algorithm for determining the physical dimensions ofthe flow stream in the detection field; algorithm for generating a datasignal corresponding to the physical dimensions of the flow stream; andinstructions for adjusting one or more parameters of the flow cytometerin response to the data signal.
 24. The system according to claim 23,wherein the flow stream in the detection stream is continuous.
 25. Thesystem according to claim 23, wherein the detection field comprises theflow stream upstream from the flow stream break-off point.
 26. Thesystem according to claim 23, wherein the physical dimension is thewidth of the flow stream.
 27. The system according to claim 26, whereinthe memory further comprises algorithm for determining the flow cellorifice diameter and generating a data signal corresponding to the flowcell nozzle orifice diameter based on the determined width of the flowstream.
 28. The system according to claim 27, wherein the memory furthercomprises algorithm for adjusting one or more parameters of the flowcytometer based on the determined flow cell nozzle orifice diameter. 29.The system according to claim 28, wherein the one or more parameters areselected from the group consisting of hydrostatic pressure, sheath fluidpressure, flow stream charge, deflection voltage, drop drive frequency,charge correction value, drop delay, drop amplitude and charge phase.30. The system according to claim 29, wherein the parameter is sheathfluid pressure.
 31. The system according to claim 29, wherein theparameter is drop drive frequency.
 32. The system according to claim 29,wherein the parameter is drop delay.